The dominance of non-halophilic archaea in autotrophic ammonia oxidation of activated sludge under salt stress: A DNA-based stable isotope probing study.

Dynamics of nitrification activity, ammonia-oxidizing archaea (AOA) and bacteria (AOB) abundance and active ammonia oxidizers of activated sludge were explored under different salinities. Results showed that specific ammonium oxidation rates were significantly negative with increasing salinity. The responses of AOA and AOB populations to salt stress were distinct. AOA abundance decreased at moderate salinities (2.5, 5 and 7 g L-1) and increased at high salinities (10, 15, 20 and 30 g L-1), while AOB abundance showed opposite tendency. DNA-based stable isotope probing assays indicated AOA exclusively dominated active ammonia oxidation of test samples under different salinities. The active AOA communities retrieved were all non-halophilic and regulated by salinities. Candidatus Nitrosocosmicus exaquare and Ca. Nitrosocosmicus franklandus were the predominantly active AOA in both salt-free and salt-containing microcosms, while 13C-labeled Nitrososphaera viennensis and Ca. Nitrososphaera gargensis were only retrieved from the microcosms amended with 0 and 30 g L-1 salinity, respectively.

More obvious air pollution impacts on variations in bacteria than fungi and their co-occurrences with ammonia-oxidizing microorganisms in PM2.5.

Based on long-term systematic sampling, information is currently limited regarding the impacts of different air pollution levels on variations of bacteria, fungi and ammonia-oxidizing microorganisms (AOMs) in fine particulate matter (PM2.5), especially their interactions. Here, PM2.5 samples were weekly collected at different air pollution levels in Beijing, China during one-year period. Microbial composition was profiled using Illumina sequencing, and their interactions were further investigated to reveal the hub genera with network analysis. Diversity of bacteria and fungi showed obvious seasonal variations, and the heavy- or severe-pollution levels mainly affected the diversity and composition of bacteria, but not fungi. While, the community structure of both bacteria and fungi was influenced by the combination of air pollution levels and seasons. The most abundant bacterial genera and some genera with highest abundance in heavy- or severe-pollution days were the hub bacteria in PM2.5. Whereas, only the dominant fungi in light-pollution days in winter were the hub fungi in PM2.5. The complex positive correlations of bacterial or fungal pathogens would aggravate the air pollution effects on human health, despite of their low relative abundances. Moreover, the strong co-occurrence and co-exclusion patterns of bacteria and fungi in PM2.5 were identified. Furthermore, the hub environmental factors (e.g., relative humidity and atmospheric pressure) may play central roles in the distributions of bacteria and fungi, including pathogens. Importantly, AOMs showed significant co-occurrence patterns with the main bacterial and fungal genera and potential pathogens, providing possible microbiological evidences for controlling ammonia emissions to effectively reduce PM2.5 pollution. These results highlighted the more obvious air pollution impacts on bacteria than fungi, and the complex bacterial-fungal interactions, as well as the important roles of AOMs in airborne microbial interactions webs, improving our understanding of bioaerosols in PM2.5.

The more important role of archaea than bacteria in nitrification of wastewater treatment plants in cold season despite their numerical relationships.

Nitrification failure of wastewater treatment plants (WWTPs) in cold season calls into investigations of the functional ammonia-oxidizing microorganisms (AOMs). In this study, we report the abundance of ammonia-oxidizing archaea (AOA), bacteria (AOB) and complete ammonia-oxidizing (comammox) Nitrospira in 23 municipal WWTPs in cold season, and explore the correlations between AOMs abundance and their relative contribution to nitrification. The copy numbers of AOA and AOB amoA gene ranged from 2.42 × 107 to 2.47 × 109 and 5.54 × 106 to 3.31 × 109 copies/g sludge, respectively. The abundance of amoA gene of Candidatus Nitrospira inopinata, an important strain of comammox Nitrospira, was stable with averaged abundance of 8.47 × 106 copies/g sludge. DNA-based stable isotope probing (DNA-SIP) assays were conducted with three typical WWTPs in which the abundance of AOA was lower than, similar to and higher than that of AOB, respectively. The results showed that considerable 13C-assimilation by AOA was detected during active nitrification in all WWTPs, whereas just a much lesser extent of 13C-incorporation by AOB and comammox Nitrospira was found in one WWTP. High-throughput sequencing with 13C-labeled DNA also showed the higher reads abundance of AOA than AOB and comammox Nitrospira. Nitrososphaera viennensis was the dominant active AOA, while Nitrosomonas oligotropha and Nitrosomonas europaea were identified as active AOB. The results obtained suggest that AOA, rather than AOB and comammox Nitrospira, dominate ammonia oxidation in WWTPs in cold season despite the numerical relationships of AOMs.

Functional genera, potential pathogens and predicted antibiotic resistance genes in 16 full-scale wastewater treatment plants treating different types of wastewater.

This study aimed to investigate the bacterial communities and antibiotic resistance genes (ARGs) in 16 wastewater treatment plants (WWTPs) treating municipal, industrial and mixed wastewater. Wastewater types showed obvious effects on bacterial communities and functions. Nitrosomonas, Nitrospira, Hyphomicrobium and Accumulibacter were the main functional genera. Mycobacterium was the dominant potential pathogens. A total of 69 ARGs were obtained, and the dominant ARGs subtypes were similar in different WWTPs. Efflux pumps were the most common resistance mechanisms. Copper and zinc resistance genes were the main metal resistance genes (MRGs). Wastewater types affected the distributions of ARGs and MRGs, and they were more similar in industrial and mixed wastewater. The co-occurrence of ARGs existed within or across ARG types, and they were also positively linked to MRGs, some functional and pathogenic genera or environmental factors. This study furthers the understanding of interactions between bacterial communities, ARGs and MRGs in different WWTPs.

[Microbial Community Dynamics During Two Sludge Granulation Processes].

Aerobic granular sludge (AGS) was cultivated in a sequencing batch reactor (SBR). In this study, AGS was broken during the formation process and then mature AGS formed again. The microbial community dynamics during two sludge granulation processes were investigated using high-throughput sequencing to reveal the dominant bacteria beneficial to AGS formation. The abundance dynamics of nitrifying microorganisms were analyzed by a quantitative polymerase chain reaction (qPCR). The results showed that the amount of extracellular protein and polysaccharides increased during two sludge granulation processes. The abundance of ammonia oxidizing archaea (AOA) increased during the first AGS formation process and during the process of AGS maturation. The abundance of ammonia oxidizing bacteria (AOB) decreased during the first AGS formation process, while it maintained a higher abundance than AOA during AGS cultivation. Microbial diversity decreased with AGS formation. The relative abundance of Proteobacteria increased by 12.29% and 5.90% during two sludge granulation processes, respectively. Candidatus Competibacter belonging to Proteobacteria was enriched during two sludge granulation processes, accounting for 14.20% in mature AGS. Overall, extracellular protein and polysaccharides may have contributed to the sludge granulation. Both AOA and AOB might have been involved in ammonia oxidation. This study indicated that Ca. Competibacter might contribute to AGS formation.

Ammonia-oxidizing bacteria dominate ammonia oxidation in a full-scale wastewater treatment plant revealed by DNA-based stable isotope probing.

A full-scale wastewater treatment plant (WWTP) with three separate treatment processes was selected to investigate the effects of seasonality and treatment process on the community structures of ammonia-oxidizing archaea (AOA) and bacteria (AOB). And then DNA-based stable isotope probing (DNA-SIP) was applied to explore the active ammonia oxidizers. The results of high-throughput sequencing indicated that treatment processes varied AOB communities rather than AOA communities. AOA slightly outnumbered AOB in most of the samples, whose abundance was significantly correlated with temperature. DNA-SIP results showed that the majority of AOB amoA gene was labeled by 13C-substrate, while just a small amount of AOA amoA gene was labeled. As revealed by high-throughput sequencing of heavy DNA, Nitrosomonadaceae-like AOB, Nitrosomonas sp. NP1, Nitrosomonas oligotropha and Nitrosomonas marina were the active AOB, and Nitrososphaera viennensis dominated the active AOA. The results indicated that AOB, not AOA, dominated active ammonia oxidation in the test WWTP.

Shifts in bacterial community composition and abundance of nitrifiers during aerobic granulation in two nitrifying sequencing batch reactors.

Shifts in bacterial community composition and abundance of nitrifiers during aerobic granulation, and the effects of wastewater composition on them were investigated using Illumina sequencing and quantitative PCR. The bacterial diversity decreased sharply during the post-granulation period. Although cultivated with different wastewater types, aerobic granular sludge (AGS) formed with similar bacterial structure. The bacterial structure in AGS was completely different from that of seed sludge. The minor genera in seed sludge, e.g., Arcobacter, Aeromonas, Flavobacterium and Acinetobacter, became the dominant genera in AGS. These genera have the potential to secrete excess extracellular polymer substances. Whereas, the dominant genera in seed sludge were found in less amount or even disappeared in AGS. During aerobic granulation, ammonia-oxidizing archaea were gradually washed-out. While, ammonia-oxidizing bacteria, complete ammonia oxidizers and nitrite-oxidizing bacteria were retained. Overall, in this study, the bacterial genera with low relative abundance in seed sludge are important for aerobic granulation.

[Short-term Effect of Roxithromycin on Abundance and Diversity of Ammonia-Oxidizing Microorganisms in Activated Sludge].

In this study, the short-term effect of roxithromycin(ROX) on the abundance and diversity of ammonia-oxidizing archaea(AOA) and ammonia-oxidizing bacteria(AOB) based on amoA gene in activated sludge were investigated by high-throughput sequencing and quantitative real-time PCR(qPCR). High-throughput sequencing overcomes the drawbacks of low sequencing depth, significant randomness and great bias of traditional Sanger sequencing. This approach can provide enough sequencing depth to comprehensively investigate the sensitive and insensitive ammonia-oxidizing microorganisms under ROX selective pressure. Lab-scale reactors were operated under ten different ROX levels. The results indicated that the environmental(0.3-30 µg·L-1) and medium(300 µg·L-1and 3000 µg·L-1) levels of ROX did not affect ammonia oxidation, while the higher concentration(5000-12000 µg·L-1) of ROX showed a significant negative effect on ammonia oxidation. The environmental and medium levels of ROX stimulated the growth of AOA, however, the higher level of ROX decreased the abundance of AOA. In addition, different levels of ROX(except 0.3 µg·L-1) caused the decrease of the abundance of AOB, which suggested that AOA was less sensitive than AOB under ROX selective pressure. The results of high-throughput sequencing showed that ROX selective pressure caused the decrease of the numbers of OTUs for AOA and increase of that for AOB. The insensitive AOA, accounting for 57.70%-97.81% of the total sequences, were Candidatus Nitrososphaera gargensis and Candidatus Nitrosoarchaeum koreensis. The insensitive AOB were Nitrosomonas oligotropha, Nitrosospira multiformis, Nitrosomonas watsonii and Nitrosomonas halophilus, accounting for 0.76%-5.10% of the total sequences. These results also indicated that AOA was insensitive to ROX, but AOB was sensitive to ROX. RDA analyses showed that AOA Ca. Nitrososphaera gargensis, Ca. Nitrosoarchaeum koreensis and AOB N. oligotropha, N. watsonii, N. halophilus were positively correlated with ROX concentrations.

[Microbial Population Dynamics During Sludge Granulation in a Simultaneous Nitrogen and Phosphorus Removal System].

In this study, domestic sewage was utilized to cultivate aerobic granular sludge (AGS) in a simultaneous nitrogen and phosphorus removal (SNPR) system. The bacterial population dynamics during the aerobic sludge granulation were investigated to reveal the granulation mechanisms using Illumina MiSeq PE300 high-throughput sequencing. Quantitative real time polymerase chain reactions (PCR) were used to investigate shifts in the abundance of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), nitrite-oxidizing bacteria (NOB) and polyphosphate accumulating organisms (PAOs). After cultivation for 100 d, the AGS was compact and demonstrated good SNPR performance. During the AGS formation process, extracellular polysaccharides obviously increased, while extracellular proteins kept relatively stable. The abundance of AOA significantly decreased during the formation of AGS process, while the abundance of PAOs increased. The bacterial diversity increased at first and then decreased during the formation of AGS. The bacterial community changed dramatically during aerobic sludge granulation. Persistent operational taxonomic units (OTUs) accounted for 92.70% of the total sequences. Proteobacteria (31.07%-53.67%), Bacteroidetes (6.70%-16.50%) and Chloroflexi (7.84%-13.36%) were the dominant phyla. Candidatus competibacter was obviously enriched in the AGS formation process (increased from 0.11% in the seed sludge to 35.33% in the AGS) and may play an important role in the formation of AGS.

Diversity, abundance and activity of ammonia-oxidizing microorganisms in fine particulate matter.

Increasing ammonia emissions could exacerbate air pollution caused by fine particulate matter (PM2.5). Therefore, it is of great importance to investigate ammonia oxidation in PM2.5. This study investigated the diversity, abundance and activity of ammonia oxidizing archaea (AOA), ammonia oxidizing bacteria (AOB) and complete ammonia oxidizers (Comammox) in PM2.5 collected in Beijing-Tianjin-Hebei megalopolis, China. Nitrosopumilus subcluster 5.2 was the most dominant AOA. Nitrosospira multiformis and Nitrosomonas aestuarii were the most dominant AOB. Comammox were present in the atmosphere, as revealed by the occurrence of Candidatus Nitrospira inopinata in PM2.5. The average cell numbers of AOA, AOB and Ca. N. inopinata were 2.82 × 104, 4.65 × 103 and 1.15 × 103 cell m-3 air, respectively. The average maximum nitrification rate of PM2.5 was 0.14 µg (NH4+-N) [m3 air·h]-1. AOA might account for most of the ammonia oxidation, followed by Comammox, while AOB were responsible for a small part of ammonia oxidation. Statistical analyses showed that Nitrososphaera subcluster 4.1 was positively correlated with organic carbon concentration, and Nitrosomonas eutropha showed positive correlation with ammonia concentration. Overall, this study expanded our knowledge concerning AOA, AOB and Comammox in PM2.5 and pointed towards an important role of AOA and Comammox in ammonia oxidation in PM2.5.

[Abundance and Community Composition of Ammonia-Oxidizing Archaea in Two Completely Autotrophic Nitrogen Removal over Nitrite Systems].

Ammonia oxidation is the first and rate-limiting step of nitrification, which was thought to be only performed by ammonia-oxidizing bacteria (AOB). In recent years, ammonia-oxidizing archaea (AOA) was also confirmed to take part in ammonia oxidation. The diversity and abundance of AOA have been investigated in various environments, however, little is known regarding the AOA in the completely autotrophic nitrogen removal over nitrite (CANON) wastewater treatment process. In this study, the abundance and diversity of AOA were investigated in the biofilm and flocculent activated sludge collected in a lab-scale (L) CANON system and a pilot-scale (P) CANON systems, respectively. The quantitative real time PCR (qPCR) was applied to investigate the abundance of AOA and the diversity of AOA was determined by polymerase chain reaction (PCR), cloning and sequencing. The qPCR results showed that the average abundance of AOA amoA gene of L and P was 2.42 x 10(6) copies x g(-1) dry sludge and 6.51 x 10(6) copies x g(-1) dry sludge, respectively. The abundance of AOA in biofilm was 10.1-14.1 times higher than that in flocculent activated sludge. For P system, the abundance of AOA in flocculent activated sludge was 1.8 times higher than that in biofilm. The results indicated that the abundance of AOA might be affected by different sludge morphology. The diversity of AOA in P system was extremely limited, only one OTU was observed, which was classified into Nitrosopumilus subcluster 5.2. The diversity of AOA in L system was higher, eight OTUs were observed, which were classified into five genera: Nitrososphaera subcluster 9, subcluster 8.1, subcluster 4.1, subcluster 1.1 and Nitrosopumilus subcluster 5.2. The diversity and abundance of AOA were different in CANON systems with different sludge morphology. AOA may play an important role in ammonia oxidation in CANON system.