Activated sludge microbial community assembly: the role of influent microbial community immigration.

Wastewater treatment plants (WWTPs) are host to diverse microbial communities and receive a constant influx of microbes from influent wastewater. However, the impact of immigrants on the structure and activities of the activated sludge (AS) microbial community remains unclear. To gain insight on this phenomenon known as perpetual community coalescence, the current study utilized controlled manipulative experiments that decoupled the influent wastewater composition from the microbial populations to reveal the fundamental mechanisms involved in immigration between sewers and AS-WWTP. The immigration dynamics of heterotrophs were analyzed by harvesting wastewater biomass solids from three different sewer systems and adding to synthetic wastewater. Immigrating influent populations were observed to contribute up to 14% of the sequencing reads in the AS. By modeling the net growth rate of taxa, it was revealed that immigrants primarily exhibited low or negative net growth rates. By developing a protocol to reproducibly grow AS-WWTP communities in the lab, we have laid down the foundational principles for the testing of operational factors creating community variations with low noise and appropriate replication. Understanding the processes that drive microbial community diversity and assembly is a key question in microbial ecology. In the future, this knowledge can be used to manipulate the structure of microbial communities and improve system performance in WWTPs.IMPORTANCEIn biological wastewater treatment processes, the microbial community composition is essential in the performance and stability of the system. This study developed a reproducible protocol to investigate the impact of influent immigration (or perpetual coalescence of the sewer and activated sludge communities) with appropriate reproducibility and controls, allowing intrinsic definitions of core and immigrant populations to be established. The method developed herein will allow sequential manipulative experiments to be performed to test specific hypothesis and optimize wastewater treatment processes to meet new treatment goals.

Accounting for the microbial assembly of each process in wastewater treatment plants (WWTPs): study of four WWTPs receiving similar influent streams.

We evaluated a unique model in which four full-scale wastewater treatment plants (WWTPs) with the same treatment schematic and fed with similar influent wastewater were tracked over an 8-month period to determine whether the community assembly would differ in the activated sludge (AS) and sand filtration (SF) stages. For each WWTP, AS and SF achieved an average of 1-log10 (90%) and <0.02-log10 (5%) reduction of total cells, respectively. Despite the removal of cells, both AS and SF had a higher alpha and beta diversity compared to the influent microbial community. Using the Sloan neutral model, it was observed that AS and SF were individually dominated by different assembly processes. Specifically, microorganisms from influent to AS were predominantly determined by the selective niche process for all WWTPs, while the microbial community in the SF was relatively favored by a stochastic, random migration process, except two WWTPs. AS also contributed more to the final effluent microbial community compared with the SF. Given that each WWTP operates the AS independently and that there is a niche selection process driven mainly by the chemical oxygen demand concentration, operational taxonomic units unique to each of the WWTPs were also identified. The findings from this study indicate that each WWTP has its distinct microbial signature and could be used for source-tracking purposes.IMPORTANCEThis study provided a novel concept that microorganisms follow a niche assembly in the activated sludge (AS) tank and that the AS contributed more than the sand filtration process toward the final microbial signature that is unique to each treatment plant. This observation highlights the importance of understanding the microbial community selected by the AS stage, which could contribute toward source-tracking the effluent from different wastewater treatment plants.

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).

Corallococcus caeni sp. nov., a novel myxobacterium isolated from activated sludge.

Two myxobacterial strains (KH5-1T and NO1) were isolated from the activated sludge tanks treating municipal sewage wastewater in Japan. These strains were recognised as myxobacteria based on their phenotypic characteristics of swarming colonies and fruiting bodies. Phylogenetic analyses using the 16S rRNA gene revealed that strains KH5-1T and NO1 were affiliated with the genus Corallococcus, with the closest neighbours being Corallococcus exercitus AB043AT (99.77% and 99.84%, respectively). Genome comparisons using orthologous average nucleotide identity (orthoANI) and digital DNA-DNA hybridisation similarity (dDDH) with strains KH5-1T and NO1 and their phylogenetically close relatives in Corallococcus spp. were below the thresholds. The major cellular fatty acids of strains KH5-1T and NO1 were iso-C15:0 (31.9%, 30.0%), summed feature 3 (comprising C16:1ω7c and/or C16:1ω6c) (20.2%, 17.7%), and iso-C17:0 (12.1%, 14.8%), and the major respiratory quinone was found to be menaquinone (MK)-8. Based on the phenotypic, chemotaxonomic, and phylogenetic evidence, strains KH5-1T and NO1 represent a new species in the genus Corallococcus, for which the proposed name is Corallococcus caeni sp. nov. The type strain is KH5-1T (= NCIMB 15510T = JCM 36609T).

Parachitinimonas caeni gen. nov., sp. nov., a novel member of family Burkholderiaceae isolated from activated sludge collected in Shenzhen, PR China.

A Gram-stain-negative, strictly aerobic and filamentous bacterial strain, designated as DQS-5T, was isolated from the activated sludge of a municipal sewage treatment plant in Shenzhen, PR China. Optimal growth was observed at 28 °C and pH 7.5. Catalase and oxidase activities were detected. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain DQS-5T was most closely related to the genera Chitinimonas and Chitinivorax (91.0-93.4 % and 92.5 % 16S rRNA gene sequence similarity, respectively) and was close to the member of the family Burkholderiaceae. The complete genome sequence of strain DQS-5T contains 5 653 844 bp and 57.3 mol% G+C. The average nucleotide identity, digital DNA-DNA hybridization and average amino acid identity values between the genome of strain DQS-5T and those of its close relatives were 75.9-77.2, 19.0-20.3 and 57.2-61.8 %, respectively. Chemotaxonomic analysis of strain DQS-5T indicated that the sole respiratory quinone was ubiquinone-8, the predominant cellular fatty acids were C16 : 0 and summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), and the major polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, aminophospholipid and aminolipid. The phylogenetic, genotypic, phenotypic and chemotaxonomic data demonstrate that strain DQS-5T represents a novel species in a novel genus within the family Burkholderiaceae, for which the name Parachitinimonas caeni gen. nov., sp. nov., is proposed. Strain DQS-5T (=KCTC 92788T=CCTCC AB 2022320T) is the type and only strain of P. caeni.

Comamonas resistens sp. nov. and Pseudomonas triclosanedens sp. nov., two members of the phylum Pseudomonadota isolated from the wastewater treatment system of a pharmaceutical factory.

Five strains of two novel species were isolated from the wastewater treatment systems of a pharmaceutical factory located in Zhejiang province, PR China. Strains ZM22T and Y6 were identified as belonging to a potential novel species of the genus Comamonas, whereas strains ZM23T, ZM24 and ZM25 were identified as belonging to a novel species of the genus Pseudomonas. These strains were characterized by polyphasic approaches including 16S rRNA gene analysis, multi-locus sequence analysis, average nucleotide identity (ANI), in silico DNA-DNA hybridization (isDDH), physiological and biochemical tests, as well as chemotaxonomic analysis. Genome-based phylogenetic analysis further confirmed that strains ZM22T and Y6 form a distinct clade closely related to Comamonas testosteroni ATCC 11996T and Comamonas thiooxydans DSM 17888T. Strains ZM23T, ZM24 and ZM25 were grouped as a separate clade closely related to Pseudomonas nitroreducens DSM 14399T and Pseudomonas nicosulfuronedens LAM1902T. The orthoANI and isDDH results indicated that strains ZM22T and Y6 belong to the same species. In addition, genomic DNA fingerprinting demonstrated that these strains do not originate from a single clone. The same results were observed for strains ZM23T, ZM24 and ZM25. Strains ZM22T and Y6 were resistant to multiple antibiotics, whereas strains ZM23T, ZM24 and ZM25 were able to degrade an emerging pollutant, triclosan. The phylogenetic, physiological and biochemical characteristics, as well as chemotaxonomy, allowed these strains to be distinguished from their genus, and we therefore propose the names Comamonas resistens sp. nov. (type strain ZM22=MCCC 1K08496T=KCTC 82561T) and Pseudomonas triclosanedens sp. nov. (type strain ZM23T=MCCC 1K08497T=JCM 36056T), respectively.

Methylosinus trichosporium OB3b bioaugmentation unleashes polyhydroxybutyrate-accumulating potential in waste-activated sludge.

BACKGROUND: Wastewater treatment plants contribute approximately 6% of anthropogenic methane emissions. Methanotrophs, capable of converting methane into polyhydroxybutyrate (PHB), offer a promising solution for utilizing methane as a carbon source, using activated sludge as a seed culture for PHB production. However, maintaining and enriching PHB-accumulating methanotrophic communities poses challenges. RESULTS: This study investigated the potential of Methylosinus trichosporium OB3b to bioaugment PHB-accumulating methanotrophic consortium within activated sludge to enhance PHB production. Waste-activated sludges with varying ratios of M. trichosporium OB3b (1:0, 1:1, 1:4, and 0:1) were cultivated. The results revealed substantial growth and methane consumption in waste-activated sludge with M. trichosporium OB3b-amended cultures, particularly in a 1:1 ratio. Enhanced PHB accumulation, reaching 37.1% in the same ratio culture, indicates the dominance of Type II methanotrophs. Quantification of methanotrophs by digital polymerase chain reaction showed gradual increases in Type II methanotrophs, correlating with increased PHB production. However, while initial bioaugmentation of M. trichosporium OB3b was observed, its presence decreased in subsequent cycles, indicating the dominance of other Type II methanotrophs. Microbial community analysis highlighted the successful enrichment of Type II methanotrophs-dominated cultures due to the addition of M. trichosporium OB3b, outcompeting Type I methanotrophs. Methylocystis and Methylophilus spp. were the most abundant in M. trichosporium OB3b-amended cultures. CONCLUSIONS: Bioaugmentation strategies, leveraging M. trichosporium OB3b could significantly enhance PHB production and foster the enrichment of PHB-accumulating methanotrophs in activated sludge. These findings contribute to integrating PHB production in wastewater treatment plants, providing a sustainable solution for resource recovery.

How Does Triclocarban Affect Sulfur Transformation in Anaerobic Systems?

Triclocarban (TCC), as a typical antimicrobial agent, accumulates at substantial levels in natural environments and engineered systems. This work investigated the impact of TCC on anaerobic sulfur transformation, especially toxic H2S production. Experimental findings revealed that TCC facilitated sulfur flow from the sludge solid phase to liquid phase, promoted sulfate reduction and sulfur-containing amino acid degradation, and largely improved anaerobic H2S production, i.e., 50-600 mg/kg total suspended solids (TSS) TCC increased the cumulative H2S yields by 24.76-478.12%. Although TCC can be partially biodegraded in anaerobic systems, the increase in H2S production can be mainly attributed to the effect of TCC rather than its degradation products. TCC was spontaneously adsorbed by protein-like substances contained in microbe extracellular polymers (EPSs), and the adsorbed TCC increased the direct electron transfer ability of EPSs, possibly due to the increase in the content of electroactive polymer protein in EPSs, the polarization of the amide group C═O bond, and the increase of the α-helical peptide dipole moment, which might be one important reason for promoting sulfur bioconversion processes. Microbial analysis showed that the presence of TCC enriched the organic substrate-degrading bacteria and sulfate-reducing bacteria and increased the abundances of functional genes encoding sulfate transport and dissimilatory sulfate reduction.

Mitigation of Triclocarban Inhibition in Microbial Electrolysis Cell-Assisted Anaerobic Digestion.

Triclocarban (TCC), as a widely used antimicrobial agent, is accumulated in waste activated sludge at a high level and inhibits the subsequent anaerobic digestion of sludge. This study, for the first time, investigated the effectiveness of microbial electrolysis cell-assisted anaerobic digestion (MEC-AD) in mitigating the inhibition of TCC to methane production. Experimental results showed that 20 mg/L TCC inhibited sludge disintegration, hydrolysis, acidogenesis, and methanogenesis processes and finally reduced methane production from traditional sludge anaerobic digestion by 19.1%. Molecular docking revealed the potential inactivation of binding of TCC to key enzymes in these processes. However, MEC-AD with 0.6 and 0.8 V external voltages achieved much higher methane production and controlled the TCC inhibition to less than 5.8%. TCC in the MEC-AD systems was adsorbed by humic substances and degraded to dichlorocarbanilide, leading to a certain detoxification effect. Methanogenic activities were increased in MEC-AD systems, accompanied by complete VFA consumption. Moreover, the applied voltage promoted cell apoptosis and sludge disintegration to release biodegradable organics. Metagenomic analysis revealed that the applied voltage increased the resistance of electrode biofilms to TCC by enriching functional microorganisms (syntrophic VFA-oxidizing and electroactive bacteria and hydrogenotrophic methanogens), acidification and methanogenesis pathways, multidrug efflux pumps, and SOS response.

Deciphering the Dual Roles of an Alginate-Based Biodegradable Flocculant in Anaerobic Fermentation of Waste Activated Sludge: Dewaterability and Degradability.

Biodegradable flocculants are rarely used in waste activated sludge (WAS) fermentation. This study introduces an alginate-based biodegradable flocculant (ABF) to enhance both the dewatering and degradation of WAS during its fermentation. Alginate was identified in structural extracellular polymeric substances (St-EPS) of WAS, with alginate-producing bacteria comprising ∼4.2% of the total bacterial population in WAS. Owing to its larger floc size, higher contact angle, and lower free energy resulting from the Lewis acid-base interaction, the addition of the prepared ABF with a network structure significantly improved the dewaterability of WAS and reduced capillary suction time (CST) by 72%. The utilization of ABF by an enriched alginate-degrading consortium (ADC) resulted in a 35.5% increase in the WAS methane yield owing to its higher hydrolytic activity on both ABF and St-EPS. Additionally, after a 30 day fermentation, CST decreased by 62% owing to the enhanced degradation of St-EPS (74.4%) and lower viscosity in the WAS + ABF + ADC group. The genus Bacteroides, comprising 12% of ADC, used alginate lyase (EC 4.2.2.3) and pectate lyase (EC 4.2.2.2 and EC 4.2.2.9) to degrade alginate and polygalacturonate in St-EPS, respectively. Therefore, this study introduces a new flocculant and elucidates its dual roles in enhancing both the dewaterability and degradability of WAS. These advancements improve WAS fermentation, resulting in higher methane production and lower CSTs.

Unveiling the Occurrence and Non-Negligible Role of Amino Sugars in Waste Activated Sludge Fermentation by an Enriched Chitin-Degradation Consortium.

Polysaccharides in extracellular polymeric substances (EPS) can form a hybrid matrix network with proteins, impeding waste-activated sludge (WAS) fermentation. Amino sugars, such as N-acetyl-d-glucosamine (GlcNAc) polymers and sialic acid, are the non-negligible components in the EPS of aerobic granules or biofilm. However, the occurrence of amino sugars in WAS and their degradation remains unclear. Thus, amino sugars (∼6.0%) in WAS were revealed, and the genera of Lactococcus and Zoogloea were identified for the first time. Chitin was used as the substrate to enrich a chitin-degrading consortium (CDC). The COD balances for methane production ranged from 83.3 and 95.1%. Chitin was gradually converted to oligosaccharides and GlcNAc after dosing with the extracellular enzyme. After doing enriched CDC in WAS, the final methane production markedly increased to 60.4 ± 0.6 mL, reflecting an increase of ∼62%. Four model substrates of amino sugars (GlcNAc and sialic acid) and polysaccharides (cellulose and dextran) could be used by CDC. Treponema (34.3%) was identified as the core bacterium via excreting chitinases (EC 3.2.1.14) and N-acetyl-glucosaminidases (EC 3.2.1.52), especially the genetic abundance of chitinases in CDC was 2.5 times higher than that of WAS. Thus, this study provides an elegant method for the utilization of amino sugar-enriched organics.

The Impact of Bisphenol A on the Anaerobic Sulfur Transformation: Promoting Sulfur Flow and Toxic H2S Production.

Bisphenol A (BPA), as a typical leachable additive from microplastics and one of the most productive bulk chemicals, is widely distributed in sediments, sewers, and wastewater treatment plants, where active sulfur cycling takes place. However, the effect of BPA on sulfur transformation, particularly toxic H2S production, has been previously overlooked. This work found that BPA at environmentally relevant levels (i.e., 50-200 mg/kg total suspended solids, TSS) promoted the release of soluble sulfur compounds and increased H2S gas production by 14.3-31.9%. The tryptophan-like proteins of microbe extracellular polymeric substances (EPSs) can spontaneously adsorb BPA, which is an enthalpy-driven reaction (ΔH = -513.5 kJ mol-1, ΔS = -1.60 kJ mol-1K -1, and ΔG = -19.52 kJ mol-1 at 35 °C). This binding changed the composition and structure of EPSs, which improved the direct electron transfer capacity of EPSs, thereby promoting the bioprocesses of organic sulfur hydrolysis and sulfate reduction. In addition, BPA presence enriched the functional microbes (e.g., Desulfovibrio and Desulfuromonas) responsible for organic sulfur mineralization and inorganic sulfate reduction and increased the abundance of related genes involved in ATP-binding cassette transporters and sulfur metabolism (e.g., Sat and AspB), which promoted anaerobic sulfur transformation. This work deepens our understanding of the interaction between BPA and sulfur transformation occurring in anaerobic environments.

Recovery of organic matters by activated sludge from municipal wastewater: Performance and characterization.

Municipal wastewater treatment processes consume a significant amount of energy and generate substantial carbon emissions. However, organic matters existing in municipal wastewater hold the potential as a valuable carbon source. Activated sludge has the potential to capture and recover the organic matters, thereby enriching carbon sources and facilitating subsequent sludge anaerobic digestion as well as in line with the concept of sustainable development. Based on above, this study investigated the enrichment and recovery characteristics and mechanisms of activated sludge adsorption on carbon sources in municipal wastewater, while optimizing the recovery conditions. The results indicated that insoluble organic matters, as well as a fraction of dissolved organic matters, can be effective recovered within approximately 40 min. Specifically, 74.1% of insoluble organic matters and 25.8% of soluble organic matters were successfully captured by the activated sludge, resulting in a 5.0% increase in sludge organic matter content. Moreover, activated sludge demonstrated remarkable recovery of particulate organic matters across various particle sizes, particularly larger particles (>5 µm) with high protein content. Notably, the dissolved biodegradable organics such as tryptophan and tyrosine protein-like substances according to 3D-EEM and lipids, proteins/amino sugars, and carbohydrates according to FT-ICR MS can be effectively recovered. Finally, the study revealed that the recovery of organic matters from the wastewater by activated sludge followed the pseudo-second-order kinetics model, with surface binding, hydrogen bonding and interparticle diffusion in sludge flocs as the primary adsorption mechanisms. This approach had abroad application prospects for improving the profitability of wastewater treatment plants.

Deep insights into the roles and microbial ecological mechanisms behind waste activated sludge digestion triggered by persulfate oxidation activated through multiple modes.

Persulfate oxidation (PS) is widely employed as a promising alternative for waste activated sludge pretreatment due to the capability of generating free radicals. The product differences and microbiological mechanisms by which PS activation triggers WAS digestion through multiple modes need to be further investigated. This study comprehensively investigated the effects of persulfate oxidation activated through multiple modes, i.e., ferrous, zero-valent iron (ZVI), ultraviolet (UV) and heat, on the performance of sludge digestion. Results showed that PS_ZVI significantly accelerated the methane production rate to 12.02 mL/g VSS. By contrast, PS_Heat promoted the sludge acidification and gained the maximum short-chain fatty acids (SCFAs) yield (277.11 ± 7.81 mg COD/g VSS), which was 3.41-fold compared to that in PS_ZVI. Moreover, ferrous and ZVI activated PS achieved the oriented conversion of acetate, the proportions of which took 73% and 78%, respectively. MiSeq sequencing results revealed that PS_Heat and PS_UV evidently enriched anaerobic fermentation bacteria (AFB) (i.e., Macellibacteroides and Clostridium XlVa). However, PS_Ferrous and PS_ZVI facilitated the enrichment of Woesearchaeota and methanogens. Furthermore, molecular ecological network and mantel test revealed the intrinsic interactions among the multiple functional microbes and environmental variables. The homo-acetogens and sulfate-reducing bacterial had potential cooperative and symbiotic relationships with AFB, while the nitrate-reducing bacteria displayed distinguishing ecological niches. Suitable activation modes for PS pretreatments resulted in an upregulation of genes expression responsible for digestion. This study established a scientific foundation for the application of sulfate radical-based oxidation on energy or high value-added chemicals recovery from waste residues.

Compatibility of vivianite-crystallization pathway of phosphorus recovery with anaerobic digestion systems of thermally hydrolyzed sludge.

Phosphorus in sewage is mostly enriched in activated sludge in wastewater treatment plants, making excess sludge an appropriate material for phosphorus recovery. The potential of vivianite (Fe3(PO4)2·8H2O) crystallization-based phosphorus recovery during the anaerobic digestion of thermally hydrolyzed sludge was discussed with influences of organic compounds on the formation of vivianite crystals being investigated in detail. Bovine serum albumin, humic acids and alginate, as model compounds of proteins, humic acids and polysaccharides, all inhibited vivianite crystallization, with the influence of humic acids being the most significant. A sludge retention time of >12 d for effective degradation of organic compounds and a certain degree of FeII excess are suggested to decrease the organics resulting inhibition. The results demonstrate the compatibility of vivianite-crystallization pathway of phosphorus recovery with anaerobic sludge digesters, and reveal the complexity of vivianite formation in the sludge with further research warranted to minimize the inhibitory influences.

Pilot-scale investigation of performance and microbial community in a novel system combining fixed and suspended activated sludge.

The conventional activated sludge (CAS) process is a widely used method for wastewater treatment due to its effectiveness and affordability. However, it can be prone to sludge abnormalities such as sludge bulking/foaming and sludge loss, which can lead to a decrease in treatment efficiency. To address these issues, a novel bag-based fixed activated sludge (BBFAS) system utilizing mesh bags to contain the sludge was developed for low carbon/nitrogen ratio wastewater treatment. Pilot-scale experiments demonstrated that the BBFAS system could successfully avoid the sludge abnormalities. Moreover, it was not affected by mass transfer resistance and exhibited significantly higher nitrogen removal efficiency, surpassing that of the CAS system by up to 78%. Additionally, the BBFAS system demonstrated comparable organic matter removal efficiency to CAS system. 16S rRNA gene high-throughput sequencing revealed that the bacterial community structure within the BBFAS system was significantly different from that of the CAS system. The bacteria associated with ammonium removal were more abundant in the BBFAS system than in the CAS system. The abundance of Nitrospira in the BBFAS could reach up to 6% and significantly higher than that in the CAS system, and they were likely responsible for both ammonia-oxidizing and nitrite-oxidizing functions. Clear stratification of microbial communities was observed from the outer to inner layers of the bag components due to the gradients of dissolved oxygen and other substrates. Overall, this study presents a promising approach for avoiding activated sludge abnormalities while maintaining high pollutant removal performance.

Soluble carbon source recovery using preconditioning coagulants for applicable short-term fermentation of waste activated sludge in WWTPs.

A huge production of waste activated sludge (WAS) has been a burden for wastewater treatment plants (WWTPs) with high disposal cost and little benefit back to wastewater purification. The short-chain fatty acids (SCFAs) produced by a short-term acidogenic fermentation of WAS before methane production have been proven to be a high-quality carbon source available for microbial denitrification process. The dual purpose of full recovery of fermentation liquid products and facilitating disposal of residual solid waste necessitate an efficient solid-liquid separation process of short-term fermentation liquid. The transformation and loss of various soluble carbon sources between solid and liquid are very important issues for carbon recovery efficiency when combining short-term fermentation and sludge dewatering in WWTPs. Here we testified the three conventional preconditioning coagulants, Polyferric Sulfate (PFS), Poly Aluminum Chloride (PAC) and Polyacrylamide (PAM), to improve the efficiency of subsequent solid-liquid separation. The results show that conversion yield of SCFAs in the liquid phase of sludge after short-term fermentation was 195 mg COD/g VSS, when using the coagulants PFS, PAC, and PAM for recovery, the recovery ratio was 79.5%, 82.0%, and 85.9%, respectively, while the dewaterability could be improved after preconditioning short-term fermentation sludge. The complexation of Al3+/Fe3+ in metal coagulants with carboxyl groups of SCFA demonstrated by Density Functional Theory calculation led to small part of soluble carbons co-migration to the solid phase, mainly a loss of high molecular weight organic compounds (carbohydrate, proteins, humic acids), while the application of PAM had little impact on carbon recovery. Economic calculations further showed PAM preconditioning short-term fermentation liquid of WAS could achieve higher recovery benefits.

A novel microbial community restructuring strategy for enhanced hydrogen production using multiple pretreatments and CSTR operation.

To achieve rapid enrichment of the targeted hydrogen-producing bacterial population and reconstruction of the microbial community in the biological hydrogen-producing reactor, the activated sludge underwent multiple pretreatments using micro-aeration, alkaline treatment, and heat treatment. The activated sludge obtained from the multiple pretreatments was inoculated into the continuous stirred tank reactor (CSTR) for continuous operations. The community structure alteration and hydrogen-producing capability of the activated sludge were analyzed throughout the operation of the reactor. We found that the primary phyla in the activated sludge population shifted to Proteobacteria, Firmicutes, and Bacteroidetes, which collectively accounted for 96.69% after undergoing several pretreatments. This suggests that the multiple pretreatments facilitated in achieving the selective enrichment of the fermentation hydrogen-producing microorganisms in the activated sludge. The CSTR start-up and continuous operation of the biological hydrogen production reactor resulted in the reactor entering a highly efficient hydrogen production stage at influent COD concentrations of 4000 mg/L and 5000 mg/L, with the highest hydrogen production rate reaching 8.19 L/d and 9.33 L/d, respectively. The main genus present during the efficient hydrogen production stage in the reactor was Ethanoligenens, accounting for up to 33% of the total population. Ethanoligenens exhibited autoaggregation capabilities and a superior capacity for hydrogen production, leading to its prevalence in the reactor and contribution to efficient hydrogen production. During high-efficiency hydrogen production, flora associated with hydrogen production exhibited up to 46.95% total relative abundance. In addition, redundancy analysis (RDA) indicated that effluent pH and COD influenced the distribution of the primary hydrogen-producing bacteria, including Ethanoligenens, Raoultella, and Pectinatus, as well as other low abundant hydrogen-producing bacteria in the activated sludge. The data indicates that the multiple pretreatments and reactor's operation has successfully enriched the hydrogen-producing genera and changed the community structure of microbial hydrogen production.

Regulatory of salinity on assembly of activated sludge microbial communities and nitrogen transformation potential in industrial plants of the lower Yangtze River basin.

This study aims to thoroughly investigate the impact mode of salinity carried by industrial wastewater on the anaerobic-anoxic-oxic (A2O) sludge in wastewater treatment plants (WWTPs). Through comprehensive investigation of the A2O stage activated sludge (AS) from 19 industrial WWTPs in the downstream area of the Yangtze River, China, A total of 38 samples of anaerobic sludge and oxic sludge were collected and analyzed. We found that salinity stress significantly inhibits the growth of the AS community, particularly evident in the anaerobic sludge community. Furthermore, the high-saline environment induces changes in the structure and functional patterns of the AS community, leading to intensive interactions and resource exchanges among microorganisms. Halophilic microorganisms may play a crucial role in this process, significantly impacting the overall community structure, especially in the oxic sludge community. Additionally, salinity stress not only suppresses the nitrogen transformation potential of the AS but also leads to the accumulation of nitrite, thereby increasing the emission potential of both NO and N2O, exacerbating the greenhouse effect of the A2O process in industrial WWTPs. The findings of this study provide necessary theoretical support for maintaining the long-term stable operation of the A2O sludge system in industrial WWTPs, reducing carbon footprint, and improving nitrogen removal efficiency.

Effects of different assembly strategies on gene annotation in activated sludge.

Activated sludge comprises diverse bacteria, fungi, and other microorganisms, featuring a rich repertoire of genes involved in antibiotic resistance, pollutant degradation, and elemental cycling. In this regard, hybrid assembly technology can revolutionize metagenomics by detecting greater gene diversity in environmental samples. Nonetheless, the optimal utilization and comparability of genomic information between hybrid assembly and short- or long-read technology remain unclear. To address this gap, we compared the performance of the hybrid assembly, short- and long-read technologies, abundance and diversity of annotated genes, and taxonomic diversity by analysing 46, 161, and 45 activated sludge metagenomic datasets, respectively. The results revealed that hybrid assembly technology exhibited the best performance, generating the most contiguous and longest contigs but with a lower proportion of high-quality metagenome-assembled genomes than short-read technology. Compared with short- or long-read technologies, hybrid assembly technology can detect a greater diversity of microbiota and antibiotic resistance genes, as well as a wider range of potential hosts. However, this approach may yield lower gene abundance and pathogen detection. Our study revealed the specific advantages and disadvantages of hybrid assembly and short- and long-read applications in wastewater treatment plants, and our approach could serve as a blueprint to be extended to terrestrial environments.