High-performance internal circulation anaerobic granular sludge reactor for cattle slaughterhouse wastewater treatment and simultaneous biogas production.

This research investigates the efficacy of a high-performance pilot-scale Internal Circulation Anaerobic Reactor inoculated with Granular Sludge (ICAGSR) for treating cattle slaughterhouse wastewater while concurrently generating biogas. The primary objective is to assess the efficiency and performance of ICAGSR in terms of organic pollutant removal and biogas production using granular anaerobic sludge. The research methodology entails operating the ICAGSR system under ambient conditions and systematically varying key parameters, including different Hydraulic Retention Times (HRTs) (24, 12, and 8 h) and Organic Loading Rates (OLRs) (3.3, 6.14, and 12.83 kg COD/m³. d). The study focuses on evaluating pollutants' removal and biogas production rates. Results reveal that the ICAGSR system achieves exceptional removal efficiency for organic pollutants, with Chemical Oxygen Demand (COD) removal exceeding 74%, 67%, and 68% at HRTs of 24, 12, and 8 h, respectively. Furthermore, the system demonstrates stable and sustainable biogas production, maintaining average methane contents of 80%, 76%, and 72% throughout the experimental period. The successful operation of the ICAGSR system underscores its potential as a viable technology for treating cattle slaughterhouse wastewater and generating renewable biogas. In conclusion, this study contributes to wastewater treatment and renewable energy production by providing a comprehensive analysis of the ICAGSR system's hydrodynamic properties. The research enhances our understanding of the system's performance optimization under varying conditions, emphasizing the benefits of utilizing ICAGSR reactors with granular sludge as an effective and sustainable approach. Identifying current gaps, future research directions aim to further refine and broaden the application of ICAGSR technology in wastewater treatment and renewable energy initiatives.

Taxonomic characterization of Sphaerotilus microaerophilus sp. nov., a sheath-forming microaerophilic bacterium of activated sludge origin.

A microaerophilic Gram-stain-negative bacilliform bacterial strain, FB-5 T, was isolated from activated sludge in Yokohama, Japan, that exhibited filamentous growth and formed a microtube (sheath). Cells were motile using a single polar flagellum. The optimum growth temperature and pH were 30 °C and 7.5, respectively. Strain FB-5 T was catalase-negative. Peptides and amino acids were utilized as energy and carbon sources. Sugars and organic acids were not utilized. Vitamin B12 enhanced the growth of strain FB-5 T. Sulfur-dependent lithotrophic growth was possible. Major respiratory quinone was UQ-8. Major fatty acids were C16:1ω7 and C16:0. The genomic DNA G + C content was 69.16%. Phylogenetic analysis of the 16S rRNA gene suggested that strain FB-5 T belongs to the genus Sphaerotilus. The close relatives were S. natans subsup. sulfidivorans and S. natans subsup. natans with 98.0% and 97.8% similarity based on the 16S rRNA gene analysis, respectively. The genome size (6.06 Mbp) was larger than that (4.39-5.07 Mbp) of the Sphaerotilus strains. The AAI values against the related strains ranged from 71.0 to 72.5%. The range of ANI values was 81.7 - 82.5%. In addition to these distinguishable features of the genome, the core genome and dDDH analyses suggested that this strain is a novel member of the genus Sphaerotilus. Based on its physiological properties and genomic features, strain FB-5 T is considered as a novel species of the genus Sphaerotilus, for which the name S. microaerophilus sp. nov. is proposed. The type strain is FB-5 T (= JCM 35424 T = KACC 23146 T).

Physicochemical parameters affecting the adhesion of ciprofloxacin-resistant Escherichia coli to activated sludge.

To investigate the physicochemical conditions necessary to stably remove antibiotic-resistant bacteria (ARB) via contact with activated sludge (AS), the adhesion of ciprofloxacin (CIP)-resistant and -susceptible Escherichia coli to AS was simulated by contact tests in the laboratory. The CIP-resistant E. coli and susceptible E. coli were removed by a 3 log smaller concentration by a 5 h contact test at maximum. Considering the hydraulic retention time of a reaction tank (∼5 h) and step-feeding operation, we considered the removal rate of E. coli in the current simulated contact test to be in agreement with the actual situation where 1-2 log concentrations of E. coli were reported to be removed from an AS reaction tank. With the increase in the AS concentration and/or dissolved oxygen, the removal rate of E. coli increased. The removal rate of CIP-resistant E. coli was greater than that of susceptible E. coli under all experimental conditions. Although the mechanism by which CIP-resistant E. coli preferably adhered to AS was not clearly understood in detail, finding optimum conditions under which bacteria, including ARB, were efficiently removed by the AS process may be possible.

Microbiome structure and function in parallel full-scale aerobic granular sludge and activated sludge processes.

Aerobic granular sludge (AGS) and conventional activated sludge (CAS) are two different biological wastewater treatment processes. AGS consists of self-immobilised microorganisms that are transformed into spherical biofilms, whereas CAS has floccular sludge of lower density. In this study, we investigated the treatment performance and microbiome dynamics of two full-scale AGS reactors and a parallel CAS system at a municipal WWTP in Sweden. Both systems produced low effluent concentrations, with some fluctuations in phosphate and nitrate mainly due to variations in organic substrate availability. The microbial diversity was slightly higher in the AGS, with different dynamics in the microbiome over time. Seasonal periodicity was observed in both sludge types, with a larger shift in the CAS microbiome compared to the AGS. Groups important for reactor function, such as ammonia-oxidising bacteria (AOB), nitrite-oxidising bacteria (NOB), polyphosphate-accumulating organisms (PAOs) and glycogen-accumulating organisms (GAOs), followed similar trends in both systems, with higher relative abundances of PAOs and GAOs in the AGS. However, microbial composition and dynamics differed between the two systems at the genus level. For instance, among PAOs, Tetrasphaera was more prevalent in the AGS, while Dechloromonas was more common in the CAS. Among NOB, Ca. Nitrotoga had a higher relative abundance in the AGS, while Nitrospira was the main nitrifier in the CAS. Furthermore, network analysis revealed the clustering of the various genera within the guilds to modules with different temporal patterns, suggesting functional redundancy in both AGS and CAS. KEY POINTS: • Microbial community succession in parallel full-scale aerobic granular sludge (AGS) and conventional activated sludge (CAS) processes. • Higher periodicity in microbial community structure in CAS compared to in AGS. • Similar functional groups between AGS and CAS but different composition and dynamics at genus level.

Wastewater treatment bacteria show differential preference for colonizing natural biopolymers.

Most reduced organic matter entering activated sludge systems is particulate (1-100-µm diameter) or colloidal (0.001-1-µm diameter), yet little is known about colonization of particulate organic matter by activated sludge bacteria. In this study, colonization of biopolymers (chitin, keratin, lignocellulose, lignin, and cellulose) by activated sludge bacteria was compared with colonization of glass beads in the presence and absence of regular nutrient amendment (acetate and ammonia). Scanning electron microscopy and quantitative PCR revealed chitin and cellulose were most readily colonized followed by lignin and lignocellulose, while keratin and glass beads were relatively resistant to colonization. Bacterial community profiles on particles compared to sludge confirmed that specific bacterial phylotypes preferentially colonize different biopolymers. Nitrifying bacteria proved adept at colonizing particles, achieving higher relative abundance on particles compared to bulk sludge. Denitrifying bacteria showed similar or lower relative abundance on particles compared to sludge. KEY POINTS: • Some activated sludge bacteria colonize natural biopolymers more readily than others. • Nitrifying bacteria are overrepresented in natural biopolymer biofilm communities. • Biopolymers in wastewater likely influence activated sludge community composition.

Detection of thermotolerant coliforms and SARS-CoV-2 RNA in sewage and recreational waters in the Ecuadorian coast: A call for improving water quality regulation.

Wastewater surveillance represents an alternative approach to regulating contamination and the early detection of infectious agents and outbreaks of diseases of public health importance. This study evaluated domestic wastewater effects on recreational waters in estuarine and seawater bodies in Guayas and Santa Elena provinces in Ecuador, South America. Fecal indicator bacteria (thermotolerant coliforms) served as key indicators for evaluation. Physical, chemical, and microbiological quality markers following the Ecuadorian environmental quality standard and the discharge of effluents to the water resource were analyzed. Samples were collected from 44 coastal sites and 2 oxidation lagoons during the dry and rainy seasons of 2020 and 2021, respectively. SARS-CoV-2 RNA was detected in samples with higher E. coli concentrations using reverse transcription quantitative PCR to detect the genes N and ORF1ab. All samples analyzed for SARS-CoV-2 showed Ct ˂ 40 for at least one gene. Four samples showed at least 20 genome copies of gene N per reaction. These were at an artisanal fishing port, an estuarine area (Palmar), a recreational bay, and an oxidation lagoon. A moderate correlation was found between SARS-CoV-2 RNA, thermotolerant coliform and E. coli (p-value ≤ 0.0037), and a strong and positive correlation between thermotolerant coliform and E. coli. (p-value ≤ 0.00001), highlighting the utility of these established parameters as a proxy of the virus. Significant differences were found in the concentrations of thermotolerant coliforms between seasons (p-value = 0.016) and sites (p-value = 0.005). The highest levels of coliforms were found in the dry season (63000 MPN/100 mL) in Anconcito and during the rainy season (14000 MPN/100 mL) at Esterillo in Playas County. It is recommended that the decentralized autonomous governments of the surveyed provinces in Ecuador implement urgent corrective actions and establish medium-term mechanisms to minimize a potential contamination route. Additional parameters must be included in the monitoring, such as Enterococcus and intestinal parasites, due to their public health implications. In the oxidation lagoons, maintenance actions must be carried out, including the dissolution of sediments, an increase in water retention times, and in situ treatment of the sludge, to improve the system's performance.

Characterization and efficacy against carbapenem-resistant Acinetobacter baumannii of a novel Friunavirus phage from sewage.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has become a new threat in recent years, owing to its rapidly increasing resistance to antibiotics and new effective therapies are needed to combat this pathogen. Phage therapy is considered to be the most promising alternative for treating CRAB infections. In this study, a novel phage, Ab_WF01, which can lyse clinical CRAB, was isolated and characterized from hospital sewage. The multiplicity of infection, morphology, one-step growth curve, stability, sensitivity, and lytic activity of the phage were also investigated. The genome of phage Ab_WF01 was 41, 317 bp in size with a GC content of 39.12% and encoded 51 open reading frames (ORFs). tRNA, virulence, and antibiotic resistance genes were not detected in the phage genome. Comparative genomic and phylogenetic analyses suggest that phage Ab_WF01 is a novel species of the genus Friunavirus, subfamily Beijerinckvirinae, and family Autographiviridae. The in vivo results showed that phage Ab_WF01 significantly increased the survival rate of CRAB-infected Galleria mellonella (from 0% to 70% at 48 h) and mice (from 0% to 60% for 7 days). Moreover, after day 3 post-infection, phage Ab_WF01 reduced inflammatory response, with strongly ameliorated histological damage and bacterial clearance in infected tissue organs (lungs, liver, and spleen) in mouse CRAB infection model. Taken together, these results show that phage Ab_WF01 holds great promise as a potential alternative agent with excellent stability for against CRAB infections.

Measuring Biomethane Potential of Food Scrap Waste Anaerobically Co-Digested with Waste-Activated Sludge Using Respirometry.

The use of respirometry to study the biokinetics of microbiota treating wastewater or digesting wastewater sludges has become more prevalent over the last few decades. The use of respirometry to examine the biokinetics of anaerobic microbiota co-digesting organic waste streams such as wastewater sludge and food scrap is an area of active research. To date, no visualized protocol has been published on the topic. Accordingly, in this protocol, we configured a respirometer to measure methane production and flow rate over time using three different food-to-microorganism (F:M) ratios and food scrap waste and waste-activated sludge as substrates. The resulting data, coupled with substrate utilization measurements, provides the basis for understanding how different substrate concentrations influence the rate at which anaerobic microbiota produce methane. Additionally, this protocol presents a method to develop biokinetic parameters (e.g., methane production rate constant and yield). Others can use this respirometry protocol to examine organic degradation under anaerobic conditions and develop microbial parameters.

Pre-exposure of Triclosan compromise tetracycline-derived antibiotic resistance in methanogenic digestion microbiome.

Triclosan (TCS) and tetracycline (TC) are commonly detected antibacterial agents in sewage and environment matrices. Nonetheless, the impact of sequential exposure to TCS and TC on the methanogenic digestion microbiome remains unknown. In this study, TCS was shown to reduce COD removal efficiency to 69.8%, but alleviated the inhibitive effect of consequent TC-amendment on the digestion microbiome. Interestingly, TCS pre-exposure resulted in abundance increase of acetotrophic Methanosaeta to 2.68%, being 2.91 folds higher than that without TCS amendment. Microbial network analyses showed that TCS pre-exposure caused microorganisms to establish a co-ecological relationship against TC disturbance. Further analyses of total antibiotic resistance genes (ARGs) showed the TCS-derived compromise of TC-induced ARGs enrichment in digestion microbiomes, e.g., 238.2% and 152.1% ARGs increase upon TC addition in digestion microbiomes without and with TCS pre-exposure, respectively. This study provides new insights into the impact of antibacterial agents on the methanogenic digestion microbiome.

A new strategy for accelerating recovery of anaerobic granular sludge after low-temperature shock: In situ regulation of quorum sensing microorganisms embedded in polyvinyl alcohol sodium alginate.

Low-temperature could inhibit the performance of anaerobic granular sludge (AnGS). Quorum sensing (QS), as a communication mode between microorganisms, can effectively regulate AnGS. In this study, a kind of embedded particles (PVA/SA@Serratia) based on signal molecule secreting bacteria was prepared by microbial immobilization technology based on polyvinyl alcohol and sodium alginate to accelerate the recovery of AnGS system after low temperature. Low-temperature shock experiment verified the positive effect of PVA/SA@Serratia on restoring the COD removal rate and methanogenesis capacity of AnGS. Further analysis by metagenomics analysis showed that PVA/SA@Serratia stimulated higher QS activity and promoted the secretion of extracellular polymeric substance (EPS) in AnGS. The rapid construction of EPS protective layer effectively accelerated the establishment of a robust microbial community structure. PVA/SA@Serratia also enhanced multiple methanogenic pathways, including direct interspecies electron transfer. In conclusion, this study demonstrated that PVA/SA@Serratia could effectively strengthen AnGS after low-temperature shock.

Feasibility of in-situ sludge fermentation coupled with partial denitrification: Key roles of initial organic matters and alkaline pH.

Achieving partial denitrification (PD) by using fermentation products extracted from waste activated sludge (WAS) rather than commercial organic matters is a promising approach for providing nitrite for anammox, while sludge reduction could also be realized by WAS reutilization. This study proposed an In-situ Sludge Fermentation coupled with Partial Denitrification (ISFPD) system and explored its performance under different conditions, including initial pH, nitrate concentrations, and organic matters. Results showed that nitrite production increased with the elevation of initial pH (from 6 to 9), and the highest nitrate-to-nitrite transformation ratio (NTR) reached 77% at initial pH 9. The PD rates and NTR were observed to be minimally influenced by initial nitrate concentrations. Acetate was preferred by denitrifying bacteria, while macromolecules such as proteins necessitated be hydrolyzed to be suitable for further utilization. The insights gained through this study paved the way for efficient nitrite production and sustainable WAS reutilization in harmony.

Conventional activated sludge vs. photo-sequencing batch reactor for enhanced nitrogen removal in municipal wastewater: Microalgal-bacterial consortium and pathogenic load insights.

Municipal wastewater treatment plants are mostly based on traditional activated sludge (AS) processes. These systems are characterised by major drawbacks: high energy consumption, large amount of excess sludge and high greenhouse gases emissions. Treatment through microalgal-bacterial consortia (MBC) is an alternative and promising solution thanks to lower energy consumption and emissions, biomass production and water sanitation. Here, microbial difference between a traditional anaerobic sludge (AS) and a consortium-based system (photo-sequencing batch reactor (PSBR)) with the same wastewater inlet were characterised through shotgun metagenomics. Stable nitrification was achieved in the PSBR ensuring ammonium removal > 95 % and significant total nitrogen removal thanks to larger flocs enhancing denitrification. The new system showed enhanced pathogen removal, a higher abundance of photosynthetic and denitrifying microorganisms with a reduced emissions potential identifying this novel PSBR as an effective alternative to AS.

Promoting bioremediation of brewery wastewater, production of bioelectricity and microbial community shift by sludge microbial fuel cells using biochar as anode.

Seeking low-cost and eco-friendly electrode catalyst of microbial fuel cell (MFC) reactor has received extensive attention in recent decades. In this study, a sludge MFC was coupled with biochar-modified-anode (BC-300, BC-400, and BC-500) for actual brewery wastewater treatment. The physicochemical properties of biochar largely depended on the pyrolysis temperature, further affecting the removal efficiency of wastewater indicators. BC-400 MFC proved to be efficient for TN and NH4+-N removal, while the maximum removal efficiencies of COD and TP were achieved by BC-500 MFC, reaching respectively 97.14 % and 89.67 %. Biochar could promote the degradation of dissolved organic matter (DOM) in wastewater by increasing the electrochemical performances of MFC. The maximum output voltage of BC-400 MFC reached 410.24 mV, and the maximum electricity generation of 108.05 mW/m2 was also obtained, surpassing the pristine MFC (BCC-MFC) by 4.67 times. High-throughput sequencing results illustrated that the enrichment of electrochemically active bacteria (EAB) and functional bacteria (Longilinea, Denitratisoma, and Pseudomonas) in BC-MFCs, contributed to pollutants degradation and electron transfer. Furthermore, biochar affected directly the electrical conductivity of wastewater, simultaneously changing microbial community composition of MFC anode. Considering both enhanced removal efficiency of pollutants and increased power generation, the results of this study would offer technical reference for the application of biochar as MFC catalyst for brewery wastewater treatment.

Identification, degradation characteristics, and application of a newly isolated pyridine-degrading Paracidovorax sp. BN6-4.

The Paracidovorax sp. BN6-4 capable of degrading high concentrations of pyridine was isolated from the coking sludge. The removal rate of BN6-4 to 1,000 mg/L pyridine during 48 h was 97.49 ±1.59%. The primary intermediate metabolites of pyridine degradation by strain BN6-4 were identified by gas chromatography-mass spectrometry (GC-MS), including N-Ethylurea, acetamidoacetaldehyde, and N-Hydroxymethylacetamide, etc. Subsequently, two different biodegradation pathways of pyridine were proposed. First, the hydroxylation of pyridine to form the intermediates pyridin-2(1H)-one and 5,6-dihydropyridine-2,5-diol, the former undergoing oxidative ring opening and the latter oxidative ring opening via N-C2 and C2-C3 ring opening to ammonia and carbon dioxide. Furthermore, the organic matter was greatly degraded by the bioremediation of real coking wastewater using BN6-4. This study enriched the microbial resource for pyridine degradation and provided new insights about the biodegradation pathway of pyridine, which is of great significance for the pyridine pollution control and coking wastewater treatment.

Description and Genomic Characteristics of Diaphorobacter limosus sp. nov., Isolated from a Sewage-Treatment Plant.

A Gram-stain-negative, rod-shaped, non-motile, catalase-positive, denitrifying bacterium, designated strain Y-1T, was isolated from an aeration tank of a sewage treatment plant in China and characterized using polyphasic taxonomic approaches. Strain Y-1T could grow at 10-37 °C (optimum 25 °C), at pH 5.0-10.0 (optimum 7.0) and in the presence of 0-3.0% (w/v) NaCl (optimum 0.5%). The phylogenetic tree based on the 16S rRNA gene sequences revealed that strain Y-1T was a member of genus Diaphorobacter, and showed the highest sequence similarities with Diaphorobacter oryzae RF3T (97.50%), Diaphorobacter nitroreducens NA10BT (97.38%) and Diaphorobacter aerolatus 8604S-37T (96.56%). In terms of carbon source utilization and enzyme activities, strain Y-1T was significantly different from its similar strains. The major respiratory quinone was Q-8, and the main polar lipid was phosphatidylethanolamine. Comparative genomic analysis of strain Y-1T and other Diaphorobacter species was conducted to explore the mechanisms underlying the differences among these strains. Strain Y-1T encoded 3957 genes, consisting of 3813 protein-coding genes and 144 RNA coding genes, and encoded 652 enzymes with 31 unique enzymes compared with other related species. The DNA G + C content was 69.95 mol%. Strain Y-1T exhibited 41.71% DNA-DNA relatedness and 95% ANIb with the most related type strains.On the basis of the evidence presented from polyphasic analysis, strain Y-1T was suggested as a novel species within the genus Diaphorobacter, for which the name Diaphorobacter limosus sp. nov. is proposed, with the type strain Y-1T (= KCTC 92852T = CCTCC AB 2023032T).

Unveiling the influence of microaeration and sludge recirculation on enhancement of pharmaceutical removal and microbial community change of the novel anaerobic baffled biofilm - membrane bioreactor in treating building wastewater.

This research aims to conduct a comparative investigation of the role played by microaeration and sludge recirculation in the novel anaerobic baffled biofilm-membrane bioreactor (AnBB-MBR) for enhancing pharmaceutical removal from building wastewater. Three AnBB-MBRs - R1: AnBB-MBR, R2: AnBB-MBR with microaeration and R3: AnBB-MBR with microaeration and sludge recirculation - were operated simultaneously to remove Ciprofloxacin (CIP), Caffeine (CAF), Sulfamethoxazole (SMX) and Diclofenac (DCF) from real building wastewater at the hydraulic retention time (HRT) of 30 h for 115 days. From the removal profiles of the targeted pharmaceuticals in the AnBB-MBRs, it was found that the fixed-film compartment (C1) could significantly reduce the targeted pharmaceuticals. The remaining pharmaceuticals were further removed with the microaeration compartment. R2 exhibited the utmost removal efficiency for CIP (78.0 %) and DCF (40.8 %), while SMX was removed most successfully by R3 (microaeration with sludge recirculation) at 91.3 %, followed by microaeration in R2 (88.5 %). For CAF, it was easily removed by all AnBB-MBR systems (>90 %). The removal mechanisms indicate that the microaeration in R2 facilitated the adsorption of CIP onto microaerobic biomass, while the enhanced biodegradation of CAF, SMX and DCF was confirmed by batch biotransformation kinetics and the adsorption isotherms of the targeted pharmaceuticals. The microbial groups involved in biodegradation of the targeted compounds under microaeration were identified as nitrogen removal microbials (Nitrosomonas, Nitrospira, Thiobacillus, and Denitratisoma) and methanotrophs (Methylosarcina, Methylocaldum, and Methylocystis). Overall, explication of the integration of AnBB-MBR with microaeration (R2) confirmed it as a prospective technology for pharmaceutical removal from building wastewater due to its energy-efficient approach characterized by minimal aeration supply.

Phages in sludge from the A/O wastewater treatment process play an important role in the transmission of ARGs.

Phages can influence the horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs) through transduction, but their profiles and effects on the transmission of ARGs are unclear, especially in complex swine sludge. In this study, we investigated the characterization of phage and ARG profiles in sludge generated from anoxic/oxic (A/O) wastewater treatment processes on swine farms using metagenomes and viromes. The results demonstrated that 205-221 subtypes of ARGs could be identified in swine sludge, among which sul1, tet(M), and floR were the dominant ARGs, indicating that sludge is an important reservoir of ARGs, especially in sludge (S) tanks. The greater abundance of mobile genetic elements (MGEs) in the S tank could significantly contribute to the greater abundance of ARGs there compared to the anoxic (A) and oxic (O) tanks (P < 0.05). However, when we compared the abundances of ARGs and MGEs in the A and O tanks, we observed opposite significant differences (P < 0.05), suggesting that MGEs are not the only factor influencing the abundance of ARGs. The high proportion of lysogenic phages in sludge from the S tank can also have a major impact on the ARG profile. Siphoviridae, Myoviridae, and Podoviridae were the dominant phage families in sludge, and a network diagram of bacteria-ARG-phages revealed that dominant phages and bacteria acted simultaneously as potential hosts for ARGs, which may have led to phage-mediated HGT of ARGs. Therefore, the risk of phage-mediated HGT of ARGs cannot be overlooked.

Comparing methanol and glycerol as carbon sources for mainstream partial denitrification/anammox in an IFAS process.

This study explored the implementation of mainstream partial denitrification with anammox (PdNA) in the second anoxic zone of a wastewater treatment process in an integrated fixed film activated sludge (IFAS) configuration. A pilot study was conducted to compare the use of methanol and glycerol as external carbon sources for an IFAS PdNA startup, with a goal to optimize nitrogen removal while minimizing carbon usage. The study also investigated the establishment of anammox bacteria on virgin carriers in IFAS reactors without the use of seeding, and it is the first IFAS PdNA startup to use methanol as an external carbon source. The establishment of anammox bacteria was confirmed in both reactors 102 days after startup. Although the glycerol-fed reactor achieved a higher steady-state maximum ammonia removal rate because of anammox bacteria (1.6 ± 0.3 g/m2/day) in comparison with the methanol-fed reactor (1.2 ± 0.2 g/m2/day), both the glycerol- and methanol-fed reactors achieved similar average in situ ammonia removal rates of 0.39 ± 0.2 g/m2/day and 0.40 ± 0.2 g/m2/day, respectively. Additionally, when the upstream ammonia versus NOx (AvN) control system maintained an ideal ratio of 0.40-0.50 g/g, the methanol-fed reactor attained a lower average effluent TIN concentration (3.50 ± 1.2 mg/L) than the glycerol-fed reactor (4.43 ± 1.6 mg/L), which was prone to elevated nitrite concentrations in the effluent. Overall, this research highlights the potential for PdNA in IFAS configurations as an efficient and cost-saving method for wastewater treatment, with methanol as a viable carbon source for the establishment of anammox bacteria. PRACTITIONER POINTS: Methanol is an effective external carbon source for an anammox startup that avoids the need for costly alternative carbon sources. The methanol-fed reactor demonstrated higher TIN removal compared with the glycerol-fed reactor because of less overproduction of nitrite. Anammox bacteria was established in an IFAS reactor without seeding and used internally stored carbon to reduce external carbon addition. Controlling the influent ammonia versus NOx (AvN) ratio between 0.40 and 0.50 g/g allowed for low and stable TIN effluent conditions.

A Metagenomic Investigation of Potential Health Risks and Element Cycling Functions of Bacteria and Viruses in Wastewater Treatment Plants.

The concentration of viruses in sewage sludge is significantly higher (10-1000-fold) than that found in natural environments, posing a potential risk for human and animal health. However, the composition of these viruses and their role in the transfer of pathogenic factors, as well as their role in the carbon, nitrogen, and phosphorus cycles remain poorly understood. In this study, we employed a shotgun metagenomic approach to investigate the pathogenic bacteria and viral composition and function in two wastewater treatment plants located on a campus. Our analysis revealed the presence of 1334 amplicon sequence variants (ASVs) across six sludge samples, with 242 ASVs (41.22% of total reads) identified as pathogenic bacteria. Arcobacter was found to be the most dominant pathogen accounting for 6.79% of total reads. The virome analysis identified 613 viral genera with Aorunvirus being the most abundant genus at 41.85%. Approximately 0.66% of these viruses were associated with human and animal diseases. More than 60% of the virome consisted of lytic phages. Host prediction analysis revealed that the phages primarily infected Lactobacillus (37.11%), Streptococcus (21.11%), and Staphylococcus (7.11%). Furthermore, our investigation revealed an abundance of auxiliary metabolic genes (AMGs) involved in carbon, nitrogen, and phosphorus cycling within the virome. We also detected a total of 113 antibiotic resistance genes (ARGs), covering major classes of antibiotics across all samples analyzed. Additionally, our findings indicated the presence of virulence factors including the clpP gene accounting for approximately 4.78%, along with toxin genes such as the RecT gene representing approximately 73.48% of all detected virulence factors and toxin genes among all samples analyzed. This study expands our understanding regarding both pathogenic bacteria and viruses present within sewage sludge while providing valuable insights into their ecological functions.

Cultivation of phosphate-accumulating biofilm: Study of the effects of acyl-homoserine lactones (AHLs) and cyclic dimeric guanosine monophosphate (c-di-GMP) on the formation of biofilm and the enhancement of phosphate metabolism capacity.

This study investigated the mechanisms of microbial growth and metabolism during biofilm cultivation in the biofilm sequencing batch reactor (BSBR) process for phosphate (P) enrichment. The results showed that the sludge discharge was key to biofilm growth, as it terminated the competition for carbon (C) source between the nascent biofilm and the activated sludge. For the tested reactor, after the sludge discharge on 18 d, P metabolism and C source utilization improved significantly, and the biofilm grew rapidly. The P concentration of the recovery liquid reached up to 157.08 mg/L, which was sufficient for further P recovery via mineralization. Meta-omics methods were used to analyze metabolic pathways and functional genes in microbial growth during biofilm cultivation. It appeared that the sludge discharge activated the key genes of P metabolism and inhibited the key genes of C metabolism, which strengthened the polyphosphate-accumulating metabolism (PAM) as a result. The sludge discharge not only changed the types of polyphosphate-accumulating organisms (PAOs) but also promoted the growth of dominant PAOs. Before the sludge discharge, the necessary metabolic abilities that were spread among different microorganisms gradually concentrated into a small number of PAOs, and after the sludge discharge, they further concentrated into Candidatus_Contendobacter (P3) and Candidatus_Accumulibacter (P17). The messenger molecule C-di-GMP, produced mostly by P3 and P17, facilitated P enrichment by regulating cellular P and C metabolism. The glycogen-accumulating organism (GAO) Candidatus_Competibacter secreted N-Acyl homoserine lactones (AHLs), which stimulated the secretion of protein in extracellular polymeric substances (EPS), thus promoting the adhesion of microorganisms to biofilm and improving P metabolism via EPS-based P adsorption. Under the combined action of the dominant GAOs and PAOs, AHLs and C-di-GMP mediated QS to promote biofilm development and P enrichment. The research provides theoretical support for the cultivation of biofilm and its wider application.