Pilot-scale demonstration of one-stage partial nitritation/anammox process to treat wastewater from a coal to ethylene glycol (CtEG) plant.

One-stage partial nitritation/anammox (PN/A) process has been recognized as a sustainable technology to treat various domestic and industrial wastewater, due to its low aeration consumption and chemical dosage. However, there is no study to investigate the feasibility of PN/A to treat coal to ethylene glycol (CtEG) wastewater yet, which contains very complex and toxic compounds including ammonium, ethylene glycol, methanol and phenolic. This study for the first time achieved stable one-stage PN/A process in a pilot-scale integrated fixed-film activated sludge (IFAS) reactor treating real wastewater produced from a CtEG plant. An average nitrogen removal efficiency of 79.5% was obtained under average nitrogen loading rate of 0.65 ± 0.09 kg N·m-3·d-1 under steady state. Moreover, the kinetic model can effectively predict the nitrogen removal rate of PN/A process. Microbial community characterization showed that ammonia oxidizing bacteria (AOB) were enriched in the flocculent sludge (12.0 ± 1.3%), while anammox bacteria (AnAOB) were primarily located in the biofilm (16.1 ± 5.6%). Meanwhile, the presence of free ammonia (FA) in conjunction with residual ammonium control could efficiently suppress the growth of NOB. Collectively, this study demonstrated the one-stage PN/A process is a promising technology to remove nitrogen from CtEG wastewater.

An emerging unrated mobile reservoir for antibiotic resistant genes: Does transportation matter to the spread.

The regional distribution of antibiotic resistance genes has been caused by the use and preference of antibiotics. Not only environmental factors, but also the population movement associated with transportation development might have had a great impact, but yet less is known regarding this issue. This research study has investigated and reported that the high-speed railway train was a possible mobile reservoir of bacteria with antibiotic resistance, based on the occurrence, diversity, and abundance of antibiotic resistant bacteria (ARB), antibiotic resistance genes (ARGs), and mobile gene elements (MGEs) in untreated train wastewater. High-throughput 16S rRNA sequencing analyses have indicated that opportunistic pathogens like Pseudomonas and Enterococcuss were the predominant bacteria in all samples, especially in cultivable multi-antibiotic resistant bacteria. The further isolated Enterococcus faecalis and Enterococcus faecium exhibited multi-antibiotic resistance ability, potentially being an indicator for disinfection proficiency. Positive correlations amongst ARGs and MGEs were observed, such as between intI1 and tetW, tetA, blaTEM, among Tn916/154 and mefA/F, qnrS, implying a broad dissemination of multi-ARGs during transportation. The study findings suggested that the high-speed railway train wastewater encompassed highly abundant antibiotic-resistant pathogens, and the wastewater discharge without effective treatment may pose severe hazards to human health and ecosystem safety.

Discarded antibiotic mycelial residues derived nitrogen-doped porous carbon for electrochemical energy storage and simultaneous reduction of antibiotic resistance genes(ARGs).

The question of how to reasonably dispose and recycle antibiotic mycelial residues (AMRs), a hazardous waste, is a critical issue. The AMRs containing nitrogen-rich organic matters shows a promising alternative feedstock of nitrogen-doped porous carbons (NPCs). Here, the NPCs with the ultrahigh surface area (2574.9 m2 g-1) were prepared by using the discarded oxytetracycline mycelial residues (OMRs) and further used as an electrode for supercapacitor. A series of experiments including scanning/transmission electron microscope, Brunauer-Emmett-Teller measurement, and electrochemical impedance spectrum revealed that the NPC-2-900 exhibited a high N content, large surface area, and high electrical conductivity. The electrochemical performance of the NPC was tested by cyclic voltammetry, galvanostatic charge/discharge cycling, and rate capability test. The optimized NPC-2-900 displayed distinguish specific capacitance (307 F g-1), cycling stability (over 95% capacitance retention after 2000 cycles even at a high current density of 20 A g-1) and superior rate performance. Of particular interest, the qPCR test indicates the ARGs were reduced in the conversion process from OMRs to NPCs.

A wireless charger powered the extracellular electron transfer for hydrogen recovery from organics.

Herein, a simple wireless charger which provided an alternative to conventional connection for delivering the electricity was employed to power the microbial electrolysis cell (MEC) for hydrogen recovery from organics. The coulombic efficiency of the wireless power transmission (WPT) was 75.37%, which was nearly similar to the average value of the conventional wired power transmission (CWPT) at the same experimental conditions (78.23%). The energy efficiency was 130.58%, it was clearly that the wireless charging (141.57%) slightly resulted in energy losing compared with conductive wire connection. The saving cost of WPT-driven MEC was a promising compensation to the energy loss according to the economic analysis of WPT, i.e., the WPT can be cost-beneficial once the distance between charger and reactor beyond a limited value. Overall, the feasibility of WPT suggests a straightforward way to construct large-scale MES with low cost and comparable performance.

Stratification of Extracellular Polymeric Substances (EPS) for Aggregated Anammox Microorganisms.

Sludge aggregation and biofilm formation are the most effective approaches to solve the washout of anammox microorganisms. In this study, the structure and composition of EPS (extracellular polymeric substances) were investigated to elucidate the factors for the anammox aggregation property. Anammox sludge taken from 18 lab-scale and pilot-scale reactors treating different types of wastewater was analyzed using EEM-PARAFAC (excitation-emission matrix and parallel factor analysis), FTIR (Fourier transform infrared spectroscopy) and real-time PCR combined with multivariate statistical analysis. The results showed that slime and TB-EPS (tightly bound EPS) were closely related with water quality and sludge morphology, and could be used as the indicators for anammox microbial survival ability and microbial aggregate morphology. Furthermore, slime secreted from anammox bacterial cells may be exhibited higher viscosity to the sludge surface and easily formed the gel network to aggregate. Large amounts of hydrophobic groups of protein in TB-EPS promoted the microbial aggregation. The mechanisms of anammox aggregation explored in this study enhanced the understanding of anammox stability in wastewater treatment processes.

Modeling optimization and evaluation of tightly bound extracellular polymeric substances extraction by sonication.

Tightly bound extracellular polymeric substances (TB-EPS) are important components of sludge, playing a crucial role in the behavior of activated sludge. They are located in the innermost layer of EPS which closely combine with the cell surface and are difficult to be extracted. To fully understand the role of TB-EPS, it is extremely important to find an appropriate TB-EPS extraction protocol, which provides maximum yields of TB-EPS under the premise of minimal contribution of cell lysis. Ultrasonic method has been widely applied for TB-EPS extraction due to its unique advantages, but no one has developed a systematic and scientifically optimized method for this protocol. In this study, a novel method based on response surface methodology (RSM) was successfully developed to optimize the conditions of TB-EPS extraction. The optimal conditions were determined at an ultrasound time of 1.00 min, ultrasonic density of 5.59 W/mL and a mixed liquor suspended solid (MLSS) of around 1700-1800 mg/L. Furthermore, combined analysis of microscopy, particle size, and excitation-emission matrix (EEM) was successfully applied to evaluate the optimal conditions. The result indicates that the optimal conditions are efficient, reliable, and reproducible which effectively solves the bottleneck problem of the conflict between cell viability and TB-EPS yield, and little effect on its chemical structure.

Real-time monitoring control of sequencing batch anammox process.

Rapid recognition and timely management of the emergent situation in wastewater treatment are crucial to maintaining the stable operation of anammox process. In this study, the feasibility of pH, conductivity (Cond) and oxidation reduction potential (ORP) profiles for monitoring and controlling anammox process for synthetic wastewater treatment was evaluated, and the practicability of the method was further verified by using real wastewater. The results showed that the characteristic values of these parameter profiles exhibited high accuracy and reproducibility in indicating the endpoint of the anammox reaction. Moreover, the positive correlations between TN removal and ΔpH, ΔCond and ΔORP were found. Nevertheless, only the slope of the Cond curve was found to be significantly linearly correlated with the specific anammox activity, which was further validated by the Haldane inhibition kinetic model, suggesting that the Cond curve can be used as an immediate feedback signal on whether anammox activity was inhibited. Overall, this study presents a fast, convenient and accurate strategy based on online real-time monitoring of instrument parameters, which was conducive to tracking the nitrogen removal process dynamics and performing the necessary operations in a timely manner, and to improving the stability of anammox process in wastewater treatment.

Effects of ammonia nitrogen and organic carbon availability on microbial community structure and ecological interactions in a full-scale partial nitritation and anammox (PN/A) system.

Nutrient availability significantly impacts microbial biosynthesis, cell growth, and cell cycle progression. In this study, a full-scale plug-flow partial nitritation/anammox (PN/A) system was used to investigate variations in the microbial community structure in both immobilized carriers and flocs, as well as a gradual decrease in nutrient availability from upstream to downstream. We found that reduced ammonia nitrogen (from 150.4 to 30.6 mg/L) and organic carbon (from 415.7 to 342.8 mg/L) availability significantly lowered microbial diversity and altered microbial communities in biofilms other than flocs from upstream to downstream. The abundance of all anammox bacteria increased by 1.97 times, from 3.25 × 1010 to 6.40 × 1010 copies per gram of wet sludge, in the biofilm core microbiome. Furthermore, from upstream to downstream, taxa with lower ribosomal RNA operon copy numbers were consistently enriched in both biofilm and floc communities, indicating that slow-growing microorganisms are more likely to be enriched in low-nutrient environments. Rare taxa with a relative abundance of less than 0.1% exhibited unique metabolic functions, including amino acid, carbohydrate, cofactor, and vitamin metabolisms, which was inferred by PICRUST2 and persisted across the nutrient gradient in both the biofilm and floc communities. Despite their low abundance, they may play important roles in mediating the stability and function of the PN/A system. Overall, the results demonstrate the impact of a naturally formed ammonia nitrogen and organic carbon gradient in a full-scale plug-flow PN/A installation on nutrient availability and its effects on microbial diversity, community composition, and microbial interactions, which expands our fundamental understanding of this energy-efficient and promising biotechnology for treating high-strength ammonium wastewater.

Morphological and Spatial Heterogeneity of Microbial Communities in Pilot-Scale Autotrophic Integrated Fixed-Film Activated Sludge System Treating Coal to Ethylene Glycol Wastewater.

The understanding of microbial compositions in different dimensions is essential to achieve the successful design and operation of the partial nitritation/anammox (PN/A) process. This study investigated the microbial communities of different sludge morphologies and spatial distribution in the one-stage PN/A process of treating real coal to ethylene glycol (CtEG) wastewater at a pilot-scale integrated fixed-film activated sludge (IFAS) reactor. The results showed that ammonia-oxidizing bacteria (AOB) was mainly distributed in flocs (13.56 ± 3.16%), whereas anammox bacteria (AnAOB) was dominated in the biofilms (17.88 ± 8.05%). Furthermore, the dominant AnAOB genus in biofilms among the first three chambers was Candidatus Brocadia (6.46 ± 2.14% to 11.82 ± 6.33%), whereas it was unexpectedly transformed to Candidatus Kuenenia (9.47 ± 1.70%) and Candidatus Anammoxoglobus (8.56 ± 4.69%) in the last chamber. This demonstrated that the niche differentiation resulting from morphological (dissolved oxygen) and spatial heterogeneity (gradient distribution of nutrients and toxins) was the main reason for dominant bacterial distribution. Overall, this study presents more comprehensive information on the heterogeneous distribution and transformation of communities in PN/A processes, providing a theoretical basis for targeted culture and selection of microbial communities in practical engineering.

Evaluation of the joint effects of Cu2+, Zn2+ and Mn2+ on completely autotrophic nitrogen-removal over nitrite (CANON) process.

The completely autotrophic nitrogen-removal over nitrite (CANON) process has merits in energy saving and consumption reducing, thus being considered as an attractive alternative over the common denitrification technology. In this study, the effects of three common heavy metals (Cu2+, Zn2+ and Mn2+) in wastewater to the CANON process were evaluated comprehensively. A central composite design with response surface methodology was utilized to investigate the joint effect of these three metal ions on the nitrogen removal performance of CANON process. In accordance with the determined optimal dosage in batch tests, four bioreactors were established with different amounts of heavy metal dosage in long-term operation, which determined the optimal concentrations for Cu2+, Zn2+ and Mn2+ to be 0.25, 0.81 and 1.00 mg/L, respectively. However, the optimal dosing level determined in batch tests showed no promotion during long-term experiment. This indicated that the actual concentration of heavy metals in bioreactors during long-term operation could be higher than expectation, leading to the difference between short-term tests and long-term experiment. The distribution of metal ions revealed that Mn2+ was mainly absorbed in anammox bacteria cells while Cu2+ and Zn2+ were mostly identified inside AOB cells. Moreover, the addition of heavy metals consistently showed positive effects for the relative abundance of AOB, while only a low level of dosage could promote the abundance of anammox bacteria. Furthermore, a mathematical model was established to simulate the CANON system considering the impacts of heavy metals, which was calibrated and validated using independent dataset in this study.

Contribution of nitrous oxide to the carbon footprint of full-scale wastewater treatment plants and mitigation strategies- a critical review.

Nitrous oxide (N2O), a potent greenhouse gas, significantly contributes to the carbon footprint of wastewater treatment plants (WWTPs) and contributes significantly to global climate change and to the deterioration of the natural environment. Our understanding of N2O generation mechanisms has significantly improved in the last decade, but the development of effective N2O emission mitigation strategies has lagged owing to the complexity of parameter regulation, substandard monitoring activities, and inadequate policy criteria. Based on critically screened published studies on N2O control in full-scale WWTPs, this review elucidates N2O generation pathway identifications and emission mechanisms and summarizes the impact of N2O on the total carbon footprint of WWTPs. In particular, a linear relationship was established between N2O emission factors and total nitrogen removal efficiencies in WWTPs located in China. Promising N2O mitigation options were proposed, which focus on optimizing operating conditions and implementation of innovative treatment processes. Furthermore, the sustainable operation of WWTPs has been anticipated to convert WWTPs into absolute greenhouse gas reducers as a result of the refinement and improvement of on-site monitoring activities, mitigation mechanisms, regulation of operational parameters, modeling, and policies.

Metagenomic prediction analysis of microbial aggregation in anammox-dominated community.

Aggregation of anammox bacteria is essential to maintain high biomass concentrations and prevent the loss of biomass in anammox processes. PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was used in this study to predict the metagenomic potentials and characterize the microbial community structure and functional features in anammox aggregates (e.g., sludge flocs, biofilms, and granules). The results showed that Candidatus Brocadia was the most dominant anammox genera in all aggregates (38.0% in flocs, 69.4% in biofilm, and 52.0% in granules) and the functional gene involved in the anammox process was detected in the highest amount in biofilms, followed by granules and flocs. Furthermore, the anammox microbial aggregation pathway was explored that anammox bacteria have strong motility and high capability for early attachment. Anammox bacteria could produce large amounts of EPS (extracellular polymeric substances) regulated by quinolone and transport to extracellular environment through type II secretion system. The strong ability of c-di-GMP (bis-(3'-5')-cyclic dimeric guanosine monophosphate) synthesis enabled a stable architectural structure of aggregation. This study elucidated the aggregation mechanism of anammox microorganisms at the genetic level to enhance the stability of anammox processes. PRACTITIONER POINTS: Candidatus Brocadia was the most dominant anammox genera in aggregates. Anammox bacteria have strong motility and high attachment capability. Anammox bacteria possess strong EPS synthesis regulated by quinolone. c-di-GMP synthesis enables a stable structure of aggregation.

The application of glycine betaine to alleviate the inhibitory effect of salinity on one-stage partial nitritation/anammox process.

One-stage partial nitritation/anammox (PN/A) has been proposed as a sustainable method for removing nitrogen from various wastewater. However, the activities of ammonium-oxidizing bacteria (AOB) and anammox bacteria are often inhibited by the exposure to salinity, thereby hindering their wide application in treating industrial wastewater with high salinity. This study reports that the addition of glycine betaine (GB), which is a compatible solute, could alleviate the inhibitory effects of salinity on both AOB and anammox, thereby improving nitrogen removal performance in a one-stage PN/A system. Short-term tests showed that with an addition of GB higher than 1 mM, the activity of AOB and anammox under salinity of 30 g/L could be increased by at least 45% and 51%, respectively. The half-inhibitory concentration of AOB and anammox rose with increasing GB concentration, with 1 mM GB being the optimal cost-effective dosage. Long-term experiments also demonstrated that 1 mM GB addition could enhance nitrogen removal performance and shorten recovery time by 42.9% under a salinity stress of 30 g/L. Collectively, GB addition was found to be a feasible and effective strategy to the counteract adverse effects of salinity on PN/A process. PRACTITIONER POINTS: Glycine betaine (GB) could improving performance of the PN/A process by alleviating the inhibitory effects of salinity on both AOB and anammox bacteria. A GB concentration of 1 mM was found to be optimum in terms of effectiveness and cost. GB addition was a feasible and effective strategy to remain stabilized in the community structure of PN/A sludge. GB could optimize the nitrogen removal performance and shorten the recovery time of PN/A process under saline stress.

Performance and microbial community dynamics relationship within a step-feed anoxic/oxic/anoxic/oxic process (SF-A/O/A/O) for coking wastewater treatment.

A step-feed anoxic/oxic/anoxic/oxic (SF-A/O/A/O) was developed and successfully applied to full-scale coking wastewater treatment. The performance and microbial community were evaluated and systematically compared with the anoxic/oxic/oxic (A/O/O) process. SF-A/OA/O process exhibited efficient removal of COD, NH4+-N, TN, phenols, and cyanide with corresponding average effluent concentrations of 317.9, 1.8, 46.2, 1.1, and 0.2 mg·L-1, respectively. In particular, the TN removal efficiency of A/O/O process was only 7.8%, with an effluent concentration of 300.6 mg·L-1. Furthermore, polycyclic aromatic hydrocarbons with high molecular weight were the dominant compounds in raw coking wastewater, which were degraded to a greater extent in SF-A/OA/O. The abundance in Thiobacillus, SM1A02, and Thauera could be the main reason why SF-A/O/A/O was superior to A/O/O in treating TN. The microbial community structure of SF-A/O/A/O was similar among stages in system (P ≥ 0.05, Welch's t-test) and was less affected by environmental factors, which may have been one of the important factors in the system's strong stability.

The effects of Fe(III) and Fe(II) on anammox process and the Fe-N metabolism.

This study aims to compare the effects of different Fe stress on anammox (anaerobic ammonium oxidation) process, therefore seven identical reactors were operated under different Fe(II)/Fe(III) concentrations. After 38 days of operation, the anammox activity was highest (10.49 ± 0.41 mg-TN/(g-VSS·h)) under conditions of 5 mg/L-Fe(II), while under 30 mg/L-Fe(III) displayed severe inhibition. The results showed that continuous addition of 30 mg/L-Fe(III) would damage the composition of EPS (extracellular polymeric substances) and make anammox bacteria more sensitive to environmental stress. While high Fe(II) concentrations could result in precipitates encasing granular sludge, affecting substrate utilization. Moreover, the results of ΔNO3--N/ΔNH4+-N indicated that Fe(II)-dependent nitrate reduction was induced in reactors added with Fe(II). OM27_clade and norank_f__Burkholderiaceae might be candidates for this process according to the correlation of genera and functional genes (based on the PICRUSt 2 functional prediction). Overall, this research is expected to provide new ideas to the effects of Fe(II)/Fe(III) on anammox and to the practical application of coupled system based on anammox in wastewater treatment.

Modeling of completely autotrophic nitrogen removal process with salt and glycine betaine addition.

The susceptibility of the completely autotrophic nitrogen removal over nitrite (CANON) process to high salinity limits its widespread application. The addition of glycine betaine (GB), a type of compatible solutes that could resist osmotic stress, could be an effective strategy to enhance the salt tolerance ability of aerobic and anaerobic ammonium oxidizing bacteria (AOB and anammox bacteria) involved in the CANON process. This study aims to make use of mathematical modeling to systematically investigate the effects of salt and GB addition on the activities of AOB and anammox bacteria and the treatment performance of the CANON process. To this end, a series of dedicated batch tests and long-term experiments for the CANON process with salt and GB additions were conducted and the data was used to calibrate and validate the model established to consider the relationships between salt and GB concentrations and bacterial growth in the CANON process. The calibrated/validated CANON process model was then applied to simulate the long-term impacts of GB addition concentration and sludge retention time (SRT) on the CANON process. The results showed that 1 mM GB addition and a SRT of 50 days would be sufficient to protect AOB and anammox bacteria under the high salinity (30 g/L NaCl) conditions studied and therefore reduce the time needed to recover the treatment performance of the CANON process from exposure to salt inhibition by 35%-40%.

Nitrogen Removal Efficiency for Pharmaceutical Wastewater with a Single-Stage Anaerobic Ammonium Oxidation Process.

A single-stage anaerobic ammonium oxidation (ANAMMOX) process with an integrated biofilm-activated sludge system was carried out in a laboratory-scale flow-through reactor (volume = 57.6 L) to treat pharmaceutical wastewater containing chlortetracycline. Partial nitrification was successfully achieved after 48 days of treatment with a nitrite accumulation of 70%. The activity of ammonia oxidizing bacteria (AOB) decreased when the chemical oxygen demand (COD) concentration of the influent was 3000 mg/L. When switching to the single-stage ANAMMOX operation, (T = 32-34 °C, DO = 0.4-0.8 mg/L, pH = 8.0-8.5), the total nitrogen (TN) removal loading rate and efficiency were 1.0 kg/m3/d and 75.2%, respectively, when the ammonium concentration of the influent was 287 ± 146 mg/L for 73 days. The findings of this study imply that single-stage ANAMMOX can achieve high nitrogen removal rates and effectively treat pharmaceutical wastewater with high concentrations of COD (1000 mg/L) and ammonium.

Using cold-adapted river-bottom sediment as seed sludge for sulfur-based autotrophic denitrification operated at mesophilic and psychrophilic temperatures.

Aiming for total nitrogen (TN) pollution control in the urbanized stream, this study proposed and verified a strategy of cultivating and acclimating sulfur-based autotrophic denitrifiers by using river-bottom sediments as seed sludge, and investigated temperature effects on sulfur-based autotrophic denitrification (SAD). With thiosulfate as an electron donor, seven SAD batch reactors were operated and studied at both 15 °C and 30 °C, to compare reactor performance and their microbial community analysis results. In the first batch, three parallel reactors (A1, A2, and A3) were operated at 30 °C for 30 days. The dynamic analysis showed that sequentially decreasing temperature to 20, 15, and 10 °C had significant adverse effects on nitrate-loading rates. In the second batch, two groups of parallel reactors were operated at 30 °C (B1 and B2) and 15 °C (C1 and C2) for 45 days. High TN removal efficiencies (>95%) were achieved in all four reactors, with comparable nitrate loading rates and less nitrite accumulation at 15 °C. High-throughput sequencing revealed that genus Thiobacillus was predominant (66.3-90.0%) in all seven reactors. However, at the operational taxonomic unit level, microbial communities at 15 °C and 30 °C were significantly different, indicating that dissimilar strains were cultivated. Our findings suggested that deliberately cultivating cold-adapted denitrifiers helps SAD to achieve high TN removal at psychrophilic temperatures and thus, is important for future applications in practical TN pollution control in urbanized streams.

pH control and microbial community analysis with HCl or CO2 addition in H2-based autotrophic denitrification.

H2-based autotrophic denitrification is promising to remove nitrate from water or wastewater lacking organic carbon sources, and pH is one of its most important process parameters. HCl and CO2 addition are known as adequate pH control methods for practical purposes. However, because of H2, added CO2 may participate in microbial metabolisms and affect denitrification mechanisms. Here, a combined micro-electrolysis and autotrophic denitrification (CEAD) reactor, in which H2 is generated based on galvanic-cell reactions between zero-valent iron and carbon, was optimized and continuously operated for 233 days by adding HCl or CO2 to control pH in the range of 7.2-8.2. Microbial communities were compared between the two pH-control methods through high-throughput sequencing of 16S rRNA, nirS, and nirK genes. Under a low COD/N ratio of 0.5 in the influent (with ∼36 mgNO3--N/L), when adding HCl, the total nitrogen (TN) removal efficiency reached 91.4% ±â€¯0.9% with a 28-h hydraulic retention time (HRT). When adding CO2, the TN removal efficiency was improved to 96.5% ±â€¯1.7% with 24-h HRT. Significant differences of 16S rRNA and nirS genes between the two pH-control stages indicated the variation of microbial communities and nirS-type denitrifiers. With HCl addition, Thiobacillus, unclassified Comamonadaceae, Arenimonas, Limnobacter, and Thermomonas, which were reported previously as likely autotrophic or heterotrophic denitrifiers, were most dominant in the biofilms. With CO2 addition, the biofilms became dominated by Anaerolineaceae and Methylocystaceae (related to organic carbon metabolism), Denitratisoma (likely heterotrophic denitrifier), and uncultured bacteria TK10 and AKYG587. The results suggest that the added CO2 not only contributed to pH control but also participated in microbial metabolisms. This study provides useful insights into microbial mechanisms and further optimization of H2-based autotrophic denitrification in water and wastewater treatment.

Minimizing extracellular DNA improves the precision of microbial community dynamic analysis in response to thermal hydrolysis.

Extracellular DNA (exDNA) can induce bias when evaluating the microbiota in wastewater treatment systems, particularly when cell lysis caused by thermal hydrolysis pretreatment (THP) releasing abundant DNA. However, the influence of such exDNA is still unknown. Accordingly, this study applied a pretreatment strategy for DNA extraction with proteinase K and DNase Ⅰ to minimize the influence of exDNA when evaluating the sludge microbiota. Lactobacillus and Peptostreptococcus were confirmed as the main THP-resistant microorganisms. Gram-positive bacteria were more resistant to THP, implying that the presence of a cell wall could promote THP resistance in bacteria. Moreover, the ability to form spores did not affect the resistance of bacteria to THP. These findings showed that resistant microbiota could be effectively evaluated by excluding exDNA, which can provide important insights into the understanding of microbiota dynamic and the effects of pretreatment on the precision of microbiota analysis in sludge.