Advanced anaerobic digestion of household food waste pretreated by in situ-produced mixed enzymes via solid-state fermentation: Feasibility and application perspectives.

Enzymatic pretreatment is an effective method which can improve the anaerobic digestion (AD) efficiency of household food waste (HFW). As an alternative to expensive commercial enzymes, mixed enzymes (MEs) produced in situ from HFW by solid-state fermentation (SSF) can greatly promote the hydrolysis rate of HFW and achieve advanced anaerobic digestion (AAD) economically sustainable. In this paper, strategies for improving the efficiency of the enzyme-production process and the abundance of MEs are briefly discussed, including SSF, fungal co-cultivation, and stepwise fermentation. The feasibility of using HFW as an applicable substrate for producing MEs (amylase, protease, and lignocellulose-degrading enzymes) and its potential advantages in HFW anaerobic digestion are comprehensively illustrated. Based on the findings, an integrated AAD process of HFW pretreated with MEs produced in situ was proposed to maximise bioenergy recovery. The mass balance results showed that the total volatile solids removal rate could reach 98.56%. Moreover, the net energy output could reach 2168.62 MJ/t HFW, which is 9.79% higher than that without in situ-produced MEs and pretreatment. Finally, perspectives for further study are presented.

A compound enzyme as an additive to a continuous anaerobic dynamic membrane bioreactor for enhanced lignocellulose removal from codigestion: Performance, membrane characteristics and microorganisms.

To explore the enzyme-enhanced strategy of a continuous anaerobic dynamic membrane reactor (AnDMBR), the anaerobic codigestion system of food waste and corn straw was first operated stably, and then the best combination of compound enzymes (laccase, endo-ß-1,4-glucanase, xylanase) was determined via a series of batch trials. The results showed that the methane yield (186.8 ± 19.9 mL/g VS) with enzyme addition was 12.2 % higher than that without enzyme addition. Furthermore, the removal rates of cellulose, hemicellulose and lignin increased by 31 %, 36 % and 78 %, respectively. In addition, dynamic membranes can form faster and more stably with enzyme addition. The addition of enzymes changed the structure of microbial communities while maintaining sufficient hydrolysis bacteria (Bacteroidetes), promoting the proliferation of Proteobacteria as a dominant strain and bringing stronger acetylation ability. In summary, the compound enzyme strengthening strategy successfully improved the methane production, dynamic membrane effect, and degradation rate of lignocellulose in AnDMBR.

Exploring the maximum nitrite production rate through the granular sludge-type reactor to match the needs of anammox process realizing efficient nitrogen removal.

The reliable and efficient nitrite production rate (NPR) through nitritation process is the prerequisite for the efficient running of subsequent processes, like the anammox process and the nitrite shunt. However, there has been scant research on stable and productive nitritation process in recent years. In this study, at a stable hydraulic retention time of 12.0 h and with precise and strict DO control, the upper limit of the NPR was initially investigated using a continuous-flow granular sludge reactor. The NPR of 1.69 kg/m3/d with a nitrite production efficiency of 81.97% was finally achieved, which set a record until now in similar research. The median sludge particle size of 270.0 µm confirmed the development of clearly defined granular sludge. The genus Nitrosomonas was the major ammonium oxidizing bacteria. In conclusion, this study provides valuable insights for the practical application of the effective nitritation process driving subsequent nitrogen removal processes.

Iron Recycle-Driven Organic Capture and Sidestream Anaerobic Membrane Bioreactor for Revolutionizing Bioenergy Generation in Municipal Wastewater Treatment.

The practicality of intensifying organic matter capture for bioenergy recovery to achieve energy-neutral municipal wastewater treatment is hindered by the lack of sustainable methods. This study developed innovative processes integrating iron recycle-driven organic capture with a sidestream anaerobic membrane bioreactor (AnMBR). Iron-assisted chemically enhanced primary treatment achieved elemental redirection with 75.2% of chemical oxygen demand (COD), 20.2% of nitrogen, and 97.4% of phosphorus captured into the sidestream process as iron-enhanced primary sludge (Fe-PS). A stable and efficient biomethanation of Fe-PS was obtained in AnMBR with a high methane yield of 224 mL/g COD. Consequently, 64.1% of the COD in Fe-PS and 48.2% of the COD in municipal wastewater were converted into bioenergy. The acidification of anaerobically digested sludge at pH = 2 achieved a high iron release efficiency of 96.1% and a sludge reduction of 29.3% in total suspended solids. Ultimately, 87.4% of iron was recycled for coagulant reuse, resulting in a theoretical 70% reduction in chemical costs. The novel system evaluation exhibited a 75.2% improvement in bioenergy recovery and an 83.3% enhancement in net energy compared to the conventional system (primary sedimentation and anaerobic digestion). This self-reliant and novel process can be applied in municipal wastewater treatment to advance energy neutrality at a lower cost.

Removal of RNA viruses from swine wastewater using anaerobic membrane bioreactor: Performance and mechanisms.

The effective removal of viruses from swine wastewater using anaerobic membrane bioreactor (AnMBR) is vital to ecological safety. However, most studies have focused only on disinfectants, whereas the capabilities of the treatment process have not been investigated. In this study, the performance and mechanism of an AnMBR in the removal of porcine hepatitis E virus (HEV), porcine kobuvirus (PKoV), porcine epidemic diarrhea virus (PEDV), and transmissible gastroenteritis coronavirus (TGEV) are systematically investigated. The results show that the AnMBR effectively removes the four viruses, with average removal efficiencies of 1.62, 3.05, 2.41, and 1.34 log for HEV, PKoV, PEDV and TGEV, respectively. Biomass adsorption contributes primarily to the total virus removal in the initial stage of reactor operation, with contributions to HEV and PKoV removal exceeding 71.7 % and 68.2 %, respectively. When the membrane is fouled, membrane rejection dominated virus removal. The membrane rejection contribution test shows the significant contribution of membrane pore foulants (23-76 %). Correlation analysis shows that the surface characteristics and size differences of the four viruses contribute primarily to their different effects on biomass adsorption and membrane rejection. This study provides technical guidance for viral removal during the treatment of high-concentration swine wastewater using an AnMBR.

A new substrate equalization method for optimizing the influent conditions and fluid flow patterns of a multifed upflow anaerobic sludge blanket reactor with mature anammox granules.

To improve nitrogen removal efficiency (NRE) and achieve homogenous distribution of anammox sludge and substrate, a new substrate equalization theory and a cumulative overload index was proposed for multifed upflow anaerobic sludge bed (MUASB) reactors with mature anammox granules. The performance and flow patterns of MUASB reactors were investigated under various influent conditions. The results showed that the nitrogen removal performance and stability of MUASB reactors could be optimized by minimizing the cumulative load. The NRE gradually increased from 83.3 ± 2.2 %, 86.8 ± 4.2 % to 89.3 ± 4.1 % and 89.7 ± 1.6 % in feeding flow tests and feeding port tests, respectively. Furthermore, the flow patterns were compared based on residence time distribution and computational fluid dynamics, indicating that a better equilibrium distribution of microorganisms and substrates could be achieved in the MUASB reactors under the lowest cumulative load. Therefore, substrate equalization theory can be used to optimize the nitrogen removal performance of MUASB reactors with low-carbon footprints.

Optimizing hydraulic retention time of high-rate activated sludge designed for potential integration with partial nitritation/anammox in municipal wastewater treatment.

The integration of high-rate activated sludge (HRAS), an effective carbon redirection technology, with partial nitritation/anammox (PN/A) is a novel AB treatment process for municipal wastewater. In this study, an airlift HRAS reactor was operated in the continuous inflow mode for 200 d at a wastewater treatment plant. The balance between potential PN/A system stability and peak HRAS performance under decreasing hydraulic retention time (HRT) was optimized. Energy consumption and recovery and CO2 emissions were calculated. The results showed that the optimal HRT suitable with the PN/A process was 3 h, achieving 2-3 g/L mixed liquor volatile suspended solid, 67.8 % chemical oxygen demand (COD) recovery, 81 % total COD removal efficiency, 2.27 ± 1.03 g COD/L/d organic loading rate, 62 % aeration reduction, and 0.24 kWh/m3 power recovery potential. Such findings hold practical value and contribute to the development of the optimal AB process capable of achieving energy autonomy and carbon neutrality.

Synergistic effects of biochar addition and filtration mode optimization on mitigating membrane fouling in high-solid anaerobic membrane bioreactors.

In this study, a high-solid anaerobic membrane bioreactor was established for treating food waste, and membrane fouling rates were regulated through multivariate modulation. The anaerobic membrane bioreactor operated stably at a high organic loading rate of 28.75 gCOD/L/d achieved a methane production rate of 8.03 ± 0.61 L/L/d. Experimental findings revealed that the most effective control of membrane fouling was achieved at a filtration- relaxation ratio (F/R) of 10/90 s. This indicates that a higher relaxation frequency provided improved the mitigation of membrane fouling. Compared with single F/R modulation, the combined modulation of biochar and F/R provided enhanced control over membrane fouling. Moreover, the addition of biochar altered the sludge properties of the reactor, thereby preventing the formation of a dense cake layer. Additionally, biochar enhanced the sheer force of the fluid on the membrane surface and facilitated the separation of pollutants during the relaxation stage, thereby contributing to improved control of membrane fouling.

Competitive bio-augmentation overcoming unusual direct inhibitor inefficacy in mainstream nitrite-oxidizing bacteria suppression: Unveiling the underpinnings in microbial and nitrogen metabolism aspects.

The long-stabilized mainstream partial nitritation/Anammox (PN/A) process continues to encounter significant challenges from nitrite-oxidizing bacteria (NOB). Therefore, this study aimed to determine an efficient, rapid, and easily implementable strategy for inhibiting NOB. A laboratory-scale reactor was operated continuously for 325 days, experiencing NOB outbreak in mainstream and recovery with simulated sidestream support. The results show that direct inhibitory strategies including intermittent aeration and approximately 35 mg/L free ammonia had unusual weak inhibitory effects on NOB activity. Subsequently, the exogenous Anammox from sidestream employed as a competitive bio-augmentation approach rapidly inhibited NOB dynamics. Evidence suggests that the damaged hydroxyapatite granules under low pH conditions might have contributed to NOB dominance by diminishing Anammox bacteria activity, thereby creating a substrate-rich environment favoring NOB survival. In contrast, the introduction of exogenous Candidatus Kuenenia facilitated the nitrogen removal efficiency from 32.5 % to over 80 %. This coincided with a decrease in the relative abundance of Nitrospira from 16.5 % to 2.7 % and NOB activity from 0.34 to 0.07 g N/(g mixed liquor volatile suspended solid)/d. Metagenomic analysis reveals a decrease in the functional potential of most nitrite transport proteins, coupled with a significant increase in eukaryotic-like serine/threonine-protein kinase involved in cellular regulation, during the Anammox activity recovery. This study's findings reveal the feasibility of the bio-augmentation based on substrate competition, wherein sidestream processes support the mainstream PN/A integration, offering significant potential for practical applications.

Dynamic pulse approach to enhancing mainstream Anammox process stability: Integrating sidestream support and tackling nitrite-oxidizing bacteria challenges.

Nitrite-oxidizing bacteria (NOB) seriously threaten the partial nitritation and Anammox (PN/A) process, hindering its mainstream application. Herein, a one-stage PN/A reactor was continuously operated for 245 days under nitrogen loading rate lifted from 0.4 g N/L/d to 0.6 g N/L/d and 0.8 g N/L/d with the nitrogen removal efficiency of 71 %, 64 %, and 41 %, respectively. Furthermore, the NOB species over time was identified as Nitrospira_sp._OLB3, exhibiting an increase of the relative abundance from 0.9 % to 4.3 %. The hydroxyapatite (HAP) granules gradually lost their microbiological function of Anammox bacteria then aged, leading to NOB dominance. Therefore, one "pulse therapy" was introduced and combined with "continuous enhancement" of Anammox sludge supported by sidestream to competitively limit the NOB dynamics. The treatment's effect persisted for around two months. The strategy that returning at least 50 % of the impaired HAP granular sludge to the sidestream for recultivation could fulfill the bottlenecks of mainstream PN/A.

Kinetic and elemental characterization of HAP-based high-rate partial nitritation/anammox system orienting stability and inorganic elemental requirements.

Anammox-based processes are attractive for biological nitrogen removal, and the combination of anammox and hydroxyapatite (HAP) is promising for the simultaneous removal of nitrogen and phosphorus from wastewater. However, the kinetics of one-stage partial nitritation/anammox (PNA) in which ammonia-oxidizing bacteria (AOB) and anammox bacteria (AnAOB) exist in a reactor are poorly understood. Moreover, inorganic elements are required to promote microbial cell synthesis and growth; therefore, monitoring of elements to prevent the limitation and inhibition of the process is critical. The minimum amounts of inorganic elements required for a one-stage PNA process and the elemental flow remain unknown. Therefore, in this study, kinetics, stoichiometry, and element flow in the long-term, high-rate, continuous, one-stage HAP-PNA process with microaerobic granular sludge at 25 °C were determined using process modeling, parameter estimation, and mass balance. The biomass elemental composition was determined to be CH2.2O0.89N0.18S0.0091, and the biomass yield (Yobs) was calculated to be 0.0805 g/g NH4+-N. Therefore, a stoichiometric reaction equation for the one-stage HAP-PNA system was also proposed. The maximum specific growth rate (µm) of AnAOB and AOB were 0.0360 and 0.0982 d-1 with doubling times of 19 and 7.1 d, respectively. Finally, the elemental requirements for stable and high-rate performance were determined using element flow analysis. These findings are essential for developing the anammox-based process in a stable and resource-efficient manner and determining engineering applicability.

Comparing the mechanisms of syntrophic volatile fatty acids oxidation and methanogenesis recovery from ammonia stress in regular and biochar-assisted anaerobic digestion: Different roads lead to the same goal.

Biochar has been recognized as a promising additive to mitigate ammonia inhibition during syntrophic methanogenesis, while the key function of biochar in this process is still in debates. This study clarified the distinct mechanisms of syntrophic volatile fatty acids -oxidizing and methanogenesis recovery from ammonia inhibition in regular and biochar-assisted anaerobic digestion. Under 5 g/L ammonia stress, adding biochar shortened the methanogenic lag time by 10.9% and dramatically accelerated the maximum methane production rate from 60.3 to 94.7 mLCH4/gVSsludge/d. A photometric analysis with a nano-WO3 probe revealed that biochar enhanced the extracellular electron transfer (EET) capacity of suspended microbes (Pearson's r = -0.98), confirming that biochar facilitated methanogenesis by boosting EET between syntrophic butyrate oxidizer and methanogens. Same linear relationship between EET capacity and methanogenic rate was not observed in the control group. Microbial community integrating functional genes prediction analysis uncovered that biochar re-shaped syntrophic partners by enriching Constridium_sensu_stricto/Syntrophomonas and Methanosarcina. The functional genes encoding Co-enzyme F420 hydrogenase and formylmethanofuran dehydrogenase were upregulated by 1.4-2.3 times, consequently enhanced the CO2-reduction methanogenesis pathway. Meanwhile, the abundances of gene encoding methylene-tetrahydrofolate transformation, a series of intermediate processes involved in acetate oxidation, in the biochar-assisted group were 28.2-63.7% higher than these in control group. Comparatively, Methanosaeta played a pivotal role driving aceticlastic methanogenesis in the control group because the abundance of gene encoding acetyl-CoA decarbonylase/synthase complex increased by 1.9 times, suggesting an aceticlastic combining H2-based syntrophic methanogenesis pathway was established in control group to resist ammonia stress. A 2nd period experiment elucidated that although depending on distinct mechanisms, the volatile fatty acid oxidizers and methanogens in both groups developed sustained and stable strategies to resist ammonia stress. These findings provided new insights to understand the distinct methanogenic recovery strategy to resist toxic stress under varied environmental conditions.

Rapid start-up of an innovative pilot-scale staged PN/A continuous process for enhanced nitrogen removal from mature landfill leachate via robust NOB elimination and efficient biomass retention.

The start-up and stable operation of partial nitritation-anammox (PN/A) treatment of mature landfill leachate (MLL) still face challenges. This study developed an innovative staged pilot-scale PN/A system to enhance nitrogen removal from MLL. The staged process included a PN unit, an anammox upflow enhanced internal circulation biofilm (UEICB) reactor, and a post-biofilm unit. Rapid start-up of the continuous flow PN process (full-concentration MLL) was achieved within 35 days by controlling dissolved oxygen and leveraging free ammonia and free nitrous acid to selectively suppress nitrite-oxidizing bacteria (NOB). The UEICB was equipped with an annular flow agitator combined with the enhanced internal circulation device of the guide tube, which achieved an efficient enrichment of Candidatus Kuenenia in the biofilm (relative abundance of 33.4 %). The nitrogen removal alliance formed by the salt-tolerant anammox bacterium (Candidatus Kuenenia) and denitrifying bacteria (unclassified SBR1031 and Denitratisoma) achieved efficient nitrogen removal of UEICB (total nitrogen removal percentage: 90.8 %) and at the same time effective treatment of the refractory organic matter (ROM). The dual membrane process of UEICB fixed biofilm combined with post-biofilm is effective in sludge retention, and can stably control the effluent suspended solids (SS) at a level of less than 5 mg/L. The post-biofilm unit ensured that effluent total nitrogen (TN) remained below the 40 mg/L discharge standard (98.5 % removal efficiency). Compared with conventional nitrification-denitrification systems, the staged PN/A process substantially reduced oxygen consumption, sludge production, CO2 emissions and carbon consumption by 22.8 %, 67.1 %, 87.1 % and 87.1 %, respectively. The 195-day stable operation marks the effective implementation of the innovative pilot-scale PN/A process in treating actual MLL. This study provides insights into strategies for rapid start-up, robust NOB suppression, and anammox biomass retention to advance the application of PN/A in high-ammonia low-carbon wastewater.

Insights into current bio-processes and future perspectives of carbon-neutral treatment of industrial organic wastewater: A critical review.

With the rise of the concept of carbon neutrality, the current wastewater treatment process of industrial organic wastewater is moving towards the goal of energy conservation and carbon emission reduction. The advantages of anaerobic digestion (AD) processes in industrial organic wastewater treatment for bio-energy recovery, which is in line with the concept of carbon neutrality. This study summarized the significance and advantages of the state-of-the-art AD processes were reviewed in detail. The application of expanded granular sludge bed (EGSB) reactors and anaerobic membrane bioreactor (AnMBR) were particularly introduced for the effective treatment of industrial organic wastewater treatment due to its remarkable prospect of engineering application for the high-strength wastewater. This study also looks forward to the optimization of the AD processes through the enhancement strategies of micro-aeration pretreatment, acidic-alkaline pretreatment, co-digestion, and biochar addition to improve the stability of the AD system and energy recovery from of industrial organic wastewater. The integration of anaerobic ammonia oxidation (Anammox) with the AD processes for the post-treatment of nitrogenous pollutants for the industrial organic wastewater is also introduced as a feasible carbon-neutral process. The combination of AnMBR and Anammox is highly recommended as a promising carbon-neutral process for the removal of both organic and inorganic pollutants from the industrial organic wastewater for future perspective. It is also suggested that the AD processes combined with biological hydrogen production, microalgae culture, bioelectrochemical technology and other bio-processes are suitable for the low-carbon treatment of industrial organic wastewater with the concept of carbon neutrality in future.

Predictive modeling based on artificial neural networks for membrane fouling in a large pilot-scale anaerobic membrane bioreactor for treating real municipal wastewater.

Membrane fouling is the primary obstacle to applying anaerobic membrane bioreactors (AnMBRs) in municipal wastewater treatment. This issue holds critical significance as efficient wastewater treatment serves as a cornerstone for achieving environmental sustainability. This study uses machine learning to predict membrane fouling, taking advantage of rapid computational and algorithmic advances. Based on the 525-day operation data of a large pilot-scale AnMBR for treating real municipal wastewater, regression prediction was realized using multilayer perceptron (MLP) and long short-term memory (LSTM) artificial neural networks under substantial variations in operating conditions. The models involved employing the organic loading rate, suspended solids concentration, protein concentration in extracellular polymeric substance (EPSp), polysaccharide concentration in EPS (EPSc), reactor temperature, and flux as input features, and transmembrane pressure as the prediction target output. Hyperparameter optimization enhanced the regression prediction accuracies, and the rationality and utility of the MLP model for predicting large-scale AnMBR membrane fouling were confirmed at global and local levels of interpretability analysis. This work not only provides a methodological advance but also underscores the importance of merging environmental engineering with computational advancements to address pressing environmental challenges.

Enzyme-enhanced acidogenic fermentation of waste activated sludge: Insights from sludge structure, interfaces, and functional microflora.

Anaerobic fermentation is widely installed to recovery valuable resources and energy as CH4 from waste activated sludge (WAS), and its implementation in developing countries is largely restricted by the slow hydrolysis, poor efficiency, and complicate inert components therein. In this study, enzyme-enhanced fermentation was conducted to improve sludge solubilization from 283 to 7728 mg COD/L and to enhance volatile fatty acids (VFAs) yield by 58.6 % as compared to the conventional fermentation. The rapid release of organic carbon species, especially for tryptophan- and tyrosine-like compounds, to outer layer of extracellular polymeric substance (EPS) occurred to reduce the structural complexity and improve the sludge biodegradability towards VFAs production. Besides, upon enzymatic pretreatment the simultaneous exposure of hydrophilic and hydrophobic groups on sludge surfaces increased the interfacial hydrophilicity. By quantitative analysis via interfacial thermodynamics and XDLVO theory, it was confirmed that the stronger hydrophilic repulsion and energy barriers in particle interface enhanced interfacial mass transfer and reactions involved in acidogenic fermentation. Meanwhile, these effects stimulate the fermentation functional microflora and predominant microorganism, and the enrichment of the hydrolytic and acid-producing bacteria in metaphase and the proliferation of acetogenic bacteria, e.g., Rubrivivax (+9.4 %), in anaphase also benefits VFAs formation. This study is practically valuable to recovery valuable VFAs as carbon sources and platform chemicals from WAS and agriculture wastes.

Comparative study of high-performance mesophilic and thermophilic anaerobic membrane bioreactors in the co-digestion of sewage sludge and food waste: Methanogenic performance and energy recovery potential.

To support smart cities in terms of waste management and bioenergy recovery, the co-digestion of sewage sludge (SeS) and food waste (FW) was conducted by the anaerobic membrane bioreactor (AnMBR) under mesophilic and thermophilic conditions in this study. The biogas production rate of the thermophilic AnMBR (ThAnMBR) at the SeS to FW ratio of 0:100, 75:25, 50:50 and 100:0 was 2.84 ± 0.21, 2.51 ± 0.26, 1.54 ± 0.26 and 1.31 ± 0.08 L-biogas/L/d, inconspicuous compared with that of the mesophilic AnMBR (MeAnMBR) at 3.00 ± 0.25, 2.46 ± 0.30, 1.63 ± 0.23 and 1.30 ± 0.17 L-biogas/L/d, respectively. The higher hydrolysis ratio and the poorer rejection efficiencies of the membrane under thermophilic conditions, resulting that the permeate COD, carbohydrate and protein of the ThAnMBR was higher than that of the MeAnMBR. The lost COD that might be converted into biogas was discharged with the permeate in the ThAnMBR, which was partly responsible for the inconspicuous methanogenic performance. Furthermore, the results of energy recovery potential assessment showed that the energy return on investment (EROI) of the MeAnMBR was 4.54, 3.81, 2.69 and 2.22 at the four SeS ratios, which was higher than that of the ThAnMBR at 3.29, 2.97, 2.02 and 1.80, respectively, indicating the advantage of the MeAnMBR over the ThAnMBR in energy recovery potential. The outcomes of this study will help to choose a more favorable temperature to co-digest SeS and FW to support the construction of smart cities.

A novel method for the whole-cell detection of environmental microorganisms using hemin and tyramide signal amplification (Hemin-TSA) with a desired fluorescent dye.

A method called hemin-tyramide signal amplification (Hemin-TSA) was developed for visualization of environmental microorganisms using hemin and tyramide signal amplification. In Hemin-TSA, hemin, which has peroxidase activity, is bound to microbial cells, and a desired fluorescent dye is deposited on the microbial cells by a hemin-catalyzed TSA reaction. The protocol was initially optimized in terms of hemin concentration, hemin binding time and repeated reaction times of TSA. Hemin-TSA showed a comparative or improved signal-to-noise ratio compared to DAPI staining. The shapes of fluorescent signals obtained from microbial cells were almost morphologically identical to those observed in phase contrast microscopy. Hemin-TSA staining provided more accurate cell counts than DAPI staining, especially for actively growing cells, for which two or three spotty DAPI signals were obtained from a single cell. In addition, microbial cells that were not detected by DAPI staining were detected by Hemin-TSA with fluorescein, which enabled us to avoid high non-specific fluorescence under UV excitation. The method developed in this study allows us to visually detect microbial cells in various environments with the characteristics of better cell morphological identification, improved enumeration accuracy and selectivity of fluorescent dyes.

The stable operation of nitritation process with the continuous granular sludge-type reactor and microbial community analysis.

The nitritation step is the fundament for the biological nitrogen removal regardless of the traditional nitrification and denitrification process, the nitrite shunt process or the anammox process. Thus, exploring the effective nitritation performance is an important aspect of biological nitrogen removal. This study explored the upper limit of nitritation rate by increasing hydraulic residence time with the well-mixed and continuous granular sludge-type reactor characterized with low complexity and easy operation. The results showed that with the nitrogen loading rate of 1.0 kg/m3/d, the nitrite production rate could reach up to 0.65 kg/m3/d with the nitrite production efficiency of 63.49%, which is remarkable compared to that in the previously similar research. The microbial analysis indicated that ammonia-oxidizing bacteria was successfully enriched (13.27%) and genus Nitrosomonas was the dominant bacteria type. Besides, the activity of ammonium oxidizing bacteria in the continuous flow reactor was higher than that of other reactor types. The growth of vorticella on the sludge was also found in the reactor. The test of specific sludge activity and the microbe analysis both indicated that the nitrite-oxidizing bacteria was well inhibited during the whole experiment, which indicated the strategy of simply adjusting the dissolved oxygen is effective for running of nitritation process. The phosphorus removal performance was also achieved with a removal efficiency of 23.53%. The functional composition of the microbial community in the samples was predicted and finally transformation mechanism of nitrogen in sludge was drawn. In sum, this study indicated the superior performance of the granule sludge-type nitritation process and give a reference for the application of biological nitrogen removal technology.

Biochar facilitates methanogens evolution by enhancing extracellular electron transfer to boost anaerobic digestion of swine manure under ammonia stress.

This study explored the mechanisms by which biochar mitigates ammonia inhibition in anaerobic digestion (AD) of swine manure. Findings show 2-8 g/L exogenous ammonia dosages gradually inhibited AD, leading to decreases in the efficiencies of hydrolysis, acidogenesis and methanogenesis by 3.4-70.8%, 6.0-82.0%, and 4.9-93.8%, respectively. However, biochar addition mitigated this inhibition and facilitated methane production. Biochar enhanced microbial activities related to electron transport and extracellular electron transfer. Moreover, biochar primarily enriched Methanosarcina, which, consequently, upregulated the genes encoding formylmethanofuran dehydrogenase and methenyltetrahydromethanopterin cyclohydrolase for the CO2-reducing methanogenesis pathway by 26.9-40.8%. It is believed that biochar mediated direct interspecies electron transfer between syntrophic partners, thereby enhancing methane production under ammonia stress. Interestingly, biochar removal did not significantly impact the AD performance of the acclimated microbial community. This indicated the pivotal role of biochar in triggering methanogen evolution to mitigate ammonia stress rather than the indispensable function after the enrichment of ammonia-resistance methanogen.