Drivers of microbial beta-diversity in wastewater treatment plants in China.

As one of the most well-documented biogeographic patterns, the distance-decay relationship provides insights into the underlying mechanisms driving biodiversity distribution. Although wastewater treatment plants (WWTPs) are well-controlled engineered ecosystems, this pattern has been seen among microbial communities in activated sludge (AS). However, little is known about the relative importance of environmental heterogeneity and dispersal limitation in shaping AS microbial community across China; especially they are related to spatial scale and organism types. Here, we assessed the distance-decay relationship based on different spatial scales and microbial phylogenetic groups by analyzing 132 activated sludge (AS) samples across China comprising 3,379,200 16S rRNA sequences. Our results indicated that the drivers of distance-decay pattern in China were scale-dependent. Microbial biogeographic patterns in WWTPs were mainly driven by dispersal limitation at both local and national scales. In contrast, conductivity, SRT, and pH played dominant roles in shaping AS microbial community compositions at the regional scale. Turnover rates and the drivers of beta-diversity also varied with microorganism populations. Moreover, a quantitative relationship between dispersal limitation ratio and AS microbial turnover rate was generated. Collectively, these results highlighted the importance of considering multiple spatial scales and micro-organism types for understanding microbial biogeography in WWTPs and provided new insights into predicting variations in AS community structure in response to environmental disturbance.

Biogeography and Assembly of Microbial Communities in Wastewater Treatment Plants in China.

Scientific understanding of microbial biogeography and assembly is lacking for activated sludge microbial communities, even though the diversity of microbial communities in wastewater treatment plants (WWTPs) is thought to have a direct influence on system performance. Here, utilizing large-scale 16S rRNA gene data generated from 211 activated sludge samples collected from 15 cities across China, we show activated sludge microbes, whose growth and metabolism can be regulated followed with the metabolic theory of ecology with an apparent Ea value (apparent activation energy) of 0.08 eV. WWTPs at a lower latitude tend to harbor a more diverse array of microorganisms. In agreement with the general understanding, the activated sludge microbial assembly was mainly driven by deterministic processes and the mean annual temperature was identified as the most important factor affecting the microbial community structure. The treatment process types with similar microbial growth types and functions had a distinct impact on the activated sludge microbial community structure only when WWTPs were located near each other and received similar influent. Overall, these findings provide us with a deeper understanding of activated sludge microbial communities from an ecological perspective. Moreover, these findings suggest that, for a given set of performance characteristics (e.g., combined nitrification, denitrification, and phosphorus removal), it may be difficult to employ common engineering levers to control additional aspects of community structure due to the influence of natural environmental factors.

Long-term occurrence, resistance risk and chaotic characteristics of antibiotic resistance genes in sludge anaerobic digestion system.

The long-term occurrence, dynamics and risk of antibiotic resistance genes (ARGs) in anaerobic digestion (AD) of excess sludge (ES) are not fully understood. Therefore, 13-month metagenomic monitoring was carried out in a full-scale AD plant. The highest ARG abundance and risk scores were observed in spring. AD achieved a 35 % removal rate for the total ARG abundance, but the risk score of AD sludge was not always lower than ES samples, because of the higher proportion of Rank I ARGs in AD sludge. ARGs showed less obvious patterns under linear models compared with microbial community, implying their chaotic dynamics, which was further confirmed by nonlinearity tests. Empirical dynamic modeling performed better than the autoregressive integrated moving average model for ARG dynamics, especially for those with simple and nonlinear dynamics. This study highlighted spring for its higher ARG abundance and risk, and recommended nonlinear models for revealing the dynamics of ARGs.

A hyperthermophilic anaerobic fermentation platform for highly efficient short chain fatty acids production from thermal hydrolyzed sludge.

In this study, a carboxylate platform of hyperthermophilic (70 â„ƒ) anaerobic fermentation (HAF) for short chain fatty acids (SCFAs) production from thermal hydrolyzed sludge (THS) was established. The long-term performance for SCFAs production and the microbial communities of this HAF under different SRTs were systematically investigated. Under the optimum SRT of 3 d, the HAF had the highest acetate production rate of 1.12 g COD/L/d which accounted for 60% in SCFAs. It also rendered a good performance in SCFAs production, with concentration, production rate and yield of 6.61 g COD/L, 1.86 g COD/L/d and 324 g COD/kg VSSin, respectively. Nearly no biogas produced from this system, which reduced the loss of carbon sources from the system. This was due to the inhibition of methanogenesis by the hyperthermophilic condition and the high content of total ammonia nitrogen (TAN) and free ammonia nitrogen (FAN). Tepidimicrobium, Bhargavaea and XBB1006 were the dominant genus-level biomarkers under the optimum SRT, which facilitated the decomposition of monosaccharides, amino acids, terpenoids and polyketides into SCFAs. This work provides an applicable anaerobic carboxylate platform for highly efficient SCFAs production from excess sludge.

Synthesis of hierarchically structured Fe3C/CNTs composites in a FeNC matrix for use as efficient ORR electrocatalysts.

Fe-N-C has a high number of FeN x active sites and has thus been regarded as a high-performance oxygen reduction reaction (ORR) catalyst, and combining Fe3C with Fe-N-C typically boosts ORR activity. However, the catalytic mechanism remains unknown, limiting further research and development. In this study, a precipitation-solvothermal process was used in conjunction with pyrolysis to produce a series of Fe-N-C catalysts derived from a zeolitic imidazolate framework (ZIF) that was composited with Fe3C. The prepared catalysts had a multiscale structure of ZIF-like carbon particles and rod-like structures, as well as bamboo-like carbon nanotubes (CNTs) and carbon layers wrapped with Fe3C particles while a series of studies revealed the origin of the rod-like structures and Fe3C phase. The hierarchical structure was beneficial to the enhanced electrocatalytic performance of catalysts for ORR. The optimal sample had the highest half-wave potential of 0.878 V vs. RHE, which was higher than that of commercial Pt/C (0.861 V vs. RHE). The ECSA of the optimal sample was 1.08 cm2 µg-1, with an electron transfer number close to 4, and functioning kinetics. The optimal sample exhibited high durability and methanol tolerance for the ORR. Finally, blocking different Fe active sites with coordination ions demonstrated that Fe(ii) was the main active site, indicating that Fe3C primarily served as a cocatalyst to optimize the electron structure of Fe-N-C, thereby synergistically improving the ORR activity.

Understanding the active sites of Fe-N-C materials and their properties in the ORR catalysis system.

Metal-N-C-based catalysts prepared by pyrolysis are frequently used in the oxygen reduction reaction (ORR). Zeolitic imidazolate frameworks (ZIFs), a type of metal organic framework (MOF), are selected as precursors due to their special structure and proper pore sizes. A series of Fe-N-C catalysts with different concentrations of 2-methylimidazole were prepared with a simple solvothermal-pyrolysis method, and the transformation productivity, morphology and ORR activity were investigated. It was found that the Fe-N-C catalyst with a 2-methylimidazole concentration of 0.53 mol L-1 had the best performance. In 0.1 M KOH solution, the half-wave potential was 0.852 V (vs. RHE), with the highest electrochemically active surface area (ECSA) of 94.1 cm2, and the ORR reaction was dominated by a 4-electron process. The current only decreased by 10.5% after 50 000 s of chronoamperometry (CA), while the half-wave potential only decreased 20 mV in 3 M methanol. Additionally, this catalyst cannot be poisoned by Cl- and SO3 2- ions in the ORR process. Finally, some typical ions including SCN-, Fe(CN)6 3- and Fe(CN)6 4- were used to inhibit the active sites, and it was determined that Fe(ii) is the real active species. The series of synthesis and testing experiments has significance in guiding optimization of the synthesis conditions and analysis of the mechanism of active sites in Fe-N-C materials.

[Impacts of F/M Ratio on Microbial Networks in Activated Sludge].

The performance of wastewater treatment plants (WWTPs) mainly relies on the microbial community in activated sludge (AS). The food to microorganisms (F/M) ratio is an important operational parameter, the recommended value of which is between 0.2 and 0.5 kg·(kg·d)-1. The F/M directly influences microbial growth and metabolism, but how the F/M ratio affects AS microbial networks and the related mechanisms remain unclear. In this study, 63 AS samples with different F/M ratios were analyzed by utilizing a random-matrix-theory based network pipeline. Firstly, all 63 samples were divided into three groups based on their F/M ratios (lower F/M group, normal F/M group, and higher F/M group). The results indicated that the lower F/M decreased microbial diversity and evenness significantly, but the higher F/M had no significant effects on the diversity of the microbial community. All three constructed networks were scale-free, small world, and modular, but network size and complexity were decreased in the lower and higher F/M groups. The network of the normal F/M ratio group was detected with the most nodes, the highest average clustering coefficient, and the shortest geodesic distance. The proportion of positive links was 76.8% when the F/M was within the normal range. On the contrary, 71.1% and 60.0% of positive links were identified when the F/M was below 0.2 and above 0.5 kg·(kg·d)-1. Moreover, 24 keystones were detected in the normal F/M network, whereas only 4 and 7 keystones were detected in the lower and higher F/M networks. Overall, our results provided clear evidence that the AS microbial community could be more stable and resilient to environmental disturbance when the F/M ratio was between 0.2 and 0.5 kg·(kg·d)-1.

Seasonal dynamics of the microbial community in two full-scale wastewater treatment plants: Diversity, composition, phylogenetic group based assembly and co-occurrence pattern.

The optimal operation and functional stability of a wastewater treatment plant (WWTP) strongly depend on the properties of its microbial community. However, a knowledge gap remains regarding the seasonal dynamics of microbial community properties, especially phylogenetic group based assembly and co-occurrence patterns. Accordingly, in this study, influent and activated sludge (AS) samples were weekly collected from 2 full-scale WWTPs for one year (89 influent and 103 AS samples in total) and examined by high-throughput Illumina-MiSeq sequencing. The results suggested that the microbial community diversity and composition in the influent fluctuated substantially with season, while those in the AS had a relatively more stable pattern throughout the year. The phylogenetic group based assembly mechanisms of AS community were identified by using "Infer Community Assembly Mechanisms by Phylogenetic-bin-based null model (iCAMP)". The results showed that drift accounted for the largest proportion (52.8%), while homogeneous selection (18.2%) was the most important deterministic process. Deterministic processes dominated in 47 microbial groups (bins), which were also found (~40%) in the AS core taxa dataset. Moreover, the results suggested that Nitrospira were more susceptible to stochastic processes in winter, which may provide a possible explanation for nitrification failure in winter. Network analysis results suggested that the network structure of the AS community could be more stable in summer and autumn. In addition, there were no identical keystone taxa found in different networks (constructed from different plants, sources, and seasons), which supported the context dependency theory. The results of this study deepened our understanding of the microbial ecology in AS systems and provided a foundation for further studies on the community regulation strategy of WWTPs.

The call for regional design code from the regional discrepancy of microbial communities in activated sludge.

Discerning the differences in activated sludge (AS) microbial community due to geographic location and environmental and operational factors is of great significance for precise design and maintenance of wastewater treatment plants (WWTPs). Hence, in this study, 150 AS samples collected from WWTPs in South China and North China were analyzed by 16 S rRNA gene sequencing. In general, AS microbial community in North China had lower diversity, higher proportions of stochastic assembly (35.7% v.s. 15.8%) and more network keystone species (19 v.s. 5) compared with southern AS community. Conductivity and SRT had significant effects on AS community in both regions. Latitude, annual mean temperature, and influent BOD, COD, and ammonia influenced South China community significantly, while pH and influent total phosphorus affected North China community. To achieve stable performance, southern WWTPs should carefully monitor fluctuations in wastewater characteristics, while northern WWTPs should monitor AS communities for shifts in the dominant taxa from immigrant strains brought in through the influent. Additionally, WWTPs in North China should be aware of the need to proactively control sludge bulking because of the high abundance and occurrence of Haliscomenobacter in these AS communities. MAIN FINDING: The call for regional design based on the regional discrepancy of microbial communities in activated sludge is uncovered and according suggestions were given.

Sludge retention time affects the microbial community structure: A large-scale sampling of aeration tanks throughout China.

Microbial communities in activated sludge (AS) have a significant influence on the functions and stability of aeration tanks in wastewater treatment plants (WWTPs). The microbial community structure is affected by various factors, among which operational parameters outcompeted as the key factors in shaping its structure. However, as an important operational parameter of aeration tank, the mechanisms by which sludge retention time (SRT) affect community properties remain unclear. In this study, 144 AS samples from aeration tanks of 48 full-scale WWTPs operating under different SRT conditions were examined via high-throughput Illumina-MiSeq sequencing technology. The results indicated that SRT significantly affected the diversity, composition, assembly, and co-occurrence patterns of the microbial community in aeration tanks. Moreover, our results provided clear evidence that the microbial communities in aeration tanks operating under SRT of 10-20 days have the highest biodiversity, the lowest stochastic processes influence, the more stable molecular ecological network structure, the lowest risks of filamentous sludge bulking and enhanced nitrogen removal potential. The microbial communities could be more stable and resilient to disturbance when aeration tanks were operated under this SRT condition. The findings of this study provided a reference for the optimization of aeration tanks from an of microbial community perspective.

Long-term performance and microbial community characteristics of pilot-scale anaerobic reactors for thermal hydrolyzed sludge digestion under mesophilic and thermophilic conditions.

Anaerobic digestion (AD) with thermal hydrolysis pretreatment has been attracted widespread attention in recent years due to its high efficiency. However, few studies focus on systematical comparison of the downstream AD processes for thermal hydrolyzed sludge and their corresponding microbial community compositions, especially on those at pilot scale and above. Thus, this study systematically compared the long-term performance and microbial communities of two pilot-scale anaerobic reactors for thermal hydrolyzed sludge digestion under mesophilic and thermophilic conditions. The results presented that mesophilic anaerobic digestion (MAD) showed a better performance of methane production than thermophilic anaerobic digestion (TAD). The hydraulic retention time (HRT) needed to be longer than 12 days in MAD while 20 days in TAD to achieve the relatively high methane production, which could be explained by that the ammonia nitrogen accumulation especially the free ammonia determined in TAD was higher than that in MAD at all HRTs, emerging an inhibition of methane yield in TAD. High-throughput Illumina sequencing results demonstrated a more diverse microbial community in MAD than that in TAD. TAD was mediated by a suite of thermophiles, such as Coprothermobacter and Methanothermobacter, while taxa harbored in MAD mostly belonged to Bacteroidetes and relatively broad types of methanogens. In addition, hydrogenotrophic methanogens were the predominant of archaea communities in both digesters probably due to the relatively high concentration of ammonia nitrogen.

Profiles of antibiotic resistance genes and virulence genes and their temporal interactions in the membrane bioreactor and oxidation ditch.

Antibiotic resistance genes (ARGs) and virulence genes (VGs) in wastewater treatment plants (WWTPs) may pose a potential threat to public health if without proper treatment. In this study, GeoChip was used to reveal the differences in ARG/VG diversity between a membrane bioreactor (MBR) and an oxidation ditch (OD) and the temporal co-occurrence patterns between ARGs and VGs. Results showed that the diversity of ARGs and VGs was lower in MBR than that in OD in the short term due to the better disinfection capability of MBR. However, the differences in diversity between two reactors disappeared in the long term because of the great variation of temperature. Instead, time-decay relationship was observed and overall turnover rate was -0.0105. Co-occurrence patterns indicate that direct connections between ARGs and VGs reduced sharply with time increasing due to the different responses of ARGs and VGs to environmental variation.

Responses of antibiotics, antibiotic resistance genes, and mobile genetic elements in sewage sludge to thermal hydrolysis pre-treatment and various anaerobic digestion conditions.

Sewage sludge from wastewater treatment plants (WWTPs) harbours large amounts of antibiotics, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs), the variation and fate of these emerging pollutants during sludge treatment processes must be thoroughly studied to reduce their potential risks to human health. In this study, 7 pilot-scale CSTR anaerobic digesters were established with the same seed sludge and fed with the same thermal hydrolysis pre-treated sewage sludge, while operating under different conditions. High-throughput quantitative PCR, UPLC-MS/MS and Illumina Hiseq-sequencing were used to systematically evaluate the responses of antibiotics, ARGs, and MGEs in sewage sludge to thermal hydrolysis pre-treatment and various anaerobic digestion (AD) conditions. The results showed that thermal hydrolysis effectively reduced the abundance (>94%) of almost all subtypes of ARGs and MGEs, and it was a powerful technology for reducing tetracyclines, macrolides, and lincosamides. Besides, the abundance of ARGs and MGEs in thermophilic digesters was lower than that in mesophilic digesters, suggesting that thermophilic digesters could be used to avoid the ARGs rebounding. In addition, the thermophilic system further reduced the concentrations of quinolones. For the digesters operated under the mesophilic conditions, a longer hydraulic retention time (HRT) facilitated the removal of antibiotics, ARGs, and MGEs. Furthermore, the microbial community and MGEs had important effects on the persistence and proliferation of ARGs in AD process. The findings of this study provide effective clues for controlling the spread of antibiotic resistance and suggest the optimal operating conditions of digesters.

[Characteristics and Regional Heterogeneity of Eukaryotic Microbial Community in Wastewater Treatment Plants].

Wastewater treatment plants (WWTPs) are important facilities to control water pollution and ensure the sustainable development of cities and humans. As an indispensable part of the activated sludge (AS) system, eukaryotic microbes play important roles in indicating the properties of AS, predicting the quality of the effluent, enhancing the purification effect, and ensuring a stable performance of the system in WWTPs. In this study, 61 AS samples from 14 full-scale WWTPs of Beijing, Shenzhen, and Wuxi were collected. Characteristics and regional heterogeneity of eukaryotic microbial community were elucidated via high-throughput sequencing (HTS) of the 18S rDNA and multi ecological and statistical methods. Results showed that eukaryotic microbial communities in different scales shared similar main members, which were mainly composed of fungi, ciliophora, and metazoa in division level with their total relative abundance up to 86.22%-89.40%. Diversity of eukaryotic microbial community in WWTPs of different cites varied. Richness and Shannon Wiener index of eukaryotic microbial communities in the AS system of Wuxi were the highest, while that of Beijing was the lowest. Diversity of eukaryotic microbes from HTS in this study was higher than that of conventional methods, but lower than the diversity of bacteria in AS systems. Regional heterogeneity of eukaryotic microbial community structure was uncovered by nonmetric multidimensional scaling based on Bray-Curtis distance and dissimilarity analysis. Results of partial mantel test and multiple regression matrix analysis showed that the eukaryotic microbial community was significantly correlated with the temperature of the aeration tank mixture and the total nitrogen concentration of the effluent of the AS system. These results help deepen the understanding of eukaryotic microbes in WWTPs.