Comprehensive analysis of effects of magnetic nanoparticles on aerobic granulation and microbial community composition: From the perspective of acyl-homoserine lactones mediated communication.

As dosing additives benefit for aerobic granular sludge (AGS) cultivation, effects of different concentrations (0, 10, 50 and 100 mg/L) of magnetic nanoparticles (Fe3O4 NPs) on aerobic granulation, contaminant removal and potential microbial community evolution related to acyl-homoserine lactones (AHLs) mediated bacterial communication were investigated with municipal wastewater. Results showed that the required time to achieve granulation ratio > 70 % was reduced by 60, 90 and 30 days in phase II with addition of 10, 50, 100 mg/L Fe3O4 NPs, respectively. 50 mg/L Fe3O4 NPs can improve contaminant removal efficiency. The promotion of relative abundance of AHLs-producing and AHLs-producing/quenching populations and AHLs-related functional genes accompanied with faster granulation. Iron-cycling-related bacteria were closely related with AHLs-related bacteria during AGS formation. Co-occurrence network analyses showed that AHLs-mediated communication may play an important role in coordinating microbial community composition and functional bacteria participating in nitrogen and polyphosphate metabolisms during aerobic granulation process.

Integrated microbial electrolysis with high-alkali pretreated sludge digestion: Insight into the effect of voltage on methanogenesis and substrate metabolism.

Integrated microbial electrolysis with anaerobic digestion is proved to be an effective way to improve methanogenesis efficiency of waste activated sludge (WAS). WAS requires pretreatment for efficient improvement of acidification or methanogenesis efficiency, but excessive acidification may inhibit the methanogenesis. In order to balance these two stages, a method for efficient WAS hydrolysis and methanogenesis has been proposed in this study by high-alkaline pretreatment integrated with microbial electrolysis system. The effects of pretreatment methods and voltage on the normal temperature digestion of WAS have also been further investigated with emphasis on the effects of voltage and substrate metabolism. The results show that compared to low-alkaline pretreatment (pH = 10), high-alkaline pretreatment (pH > 14) can double the SCOD release and promote the VFAs accumulation to 5657 ± 392 mg COD/L, but inhibit the methanogenesis process. Microbial electrolysis can alleviate this inhibition effectively through the rapid consumption of VFAs and speeding up of the methanogenesis process. The optimal methane yield of the integrated system is 120.4 ± 8.4 mL/g VSS at the voltage of 0.5 V. Enzyme activities, high-throughput and gene function prediction analysis reveal that the cathode and anode maintain the activity of methanogens under high substrate concentrations. Voltage positively responded to improved methane yield from 0.3 to 0.8 V, but higher than 1.1 V is found to be unfavorable for cathodic methanogenesis and results in additional power loss. These findings provide a perspective idea for rapid and maximum biogas recovery from WAS.

Downregulation of AC092894.1 promotes oxaliplatin resistance in colorectal cancer via the USP3/AR/RASGRP3 axis.

BACKGROUND: Oxaliplatin resistance is a complex process and has been one of the most disadvantageous factors and indeed a confrontation in the procedure of colorectal cancer. Recently, long non-coding RNAs (lncRNAs) have emerged as novel molecules for the treatment of chemoresistance, but the specific molecular mechanisms mediated by them are poorly understood. METHODS: The lncRNAs associated with oxaliplatin resistance were screened by microarray. lncRNA effects on oxaliplatin chemoresistance were then verified by gain- and loss-of-function experiments. Finally, the potential mechanism of AC092894.1 was explored by RNA pull-down, RIP, and Co-IP experiments. RESULTS: AC092894.1 representation has been demonstrated to be drastically downregulated throughout oxaliplatin-induced drug-resistant CRC cells. In vivo and in vitro experiments revealed that AC092894.1 functions to reverse chemoresistance. Studies on the mechanism suggested that AC092894.1 served as a scaffold molecule that mediated the de-ubiquitination of AR through USP3, thereby increasing the transcription of RASGRP3. Finally, sustained activation of the MAPK signaling pathway induced apoptosis in CRC cells. CONCLUSIONS: In conclusion, this study identified AC092894.1 as a suppressor of CRC chemoresistance and revealed the idea that targeting the AC092894.1/USP3/AR/RASGRP3 signaling axis is a novel option for the treatment of oxaliplatin resistance.

Multi-omics Analysis of the Role of PHGDH in Colon Cancer.

Objectives: Serine metabolism is essential for tumor cells. Endogenous serine arises from de novo synthesis pathways. As the rate-limiting enzyme of this pathway, PHGDH is highly expressed in a variety of tumors including colon cancer. Therefore, targeted inhibition of PHGDH is an important strategy for anti-tumor therapy research. However, the specific gene expression and metabolic pathways regulated by PHGDH in colon cancer are still unclear. Our study was aimed to clarified the role of PHGDH in serine metabolism in colon cancer to provide new knowledge for in-depth understanding of serine metabolism and PHGDH function in colon cancer. Methods: In this study, we analyzed the gene expression and metabolic remodeling process of colon cancer cells (SW620) after targeted inhibition of PHGDH by gene transcriptomics and metabolomics. LC-MS analysis was performed in 293T cells to PHGDH gene transcription and protein post-translational modification under depriving exogenous serine. Results: We found that amino acid transporters, amino acid metabolism, lipid synthesis related pathways compensation and other processes are involved in the response process after PHGDH inhibition. And ATF4 mediated the transcriptional expression of PHGDH under exogenous serine deficiency conditions. While LC-MS analysis of post-translational modification revealed that PHGDH produced changes in acetylation sites after serine deprivation that the K289 site was lost, and a new acetylation site K21was produced. Conclusion: Our study performed transcriptomic and metabolomic analysis by inhibiting PHGDH, thus clarifying the role of PHGDH in gene transcription and metabolism in colon cancer cells. The mechanism of high PHGDH expression in colon cancer cells and the acetylation modification that occurs in PHGDH protein were also clarified by serine deprivation. In our study, the role of PHGDH in serine metabolism in colon cancer was clarified by multi-omics analysis to provide new knowledge for in-depth understanding of serine metabolism and PHGDH function in colon cancer.

The Global mRNA Expression Profiles of Inhibiting PHGDH Induced Cisplatin Resistance in Gastric Cancer.

Objective: Drug resistance is the main hindrance to improve the prognosis of patients with gastric cancer. Amino
acid metabolic reprograming is essential to satisfy the different requirements of cancer cells during drug resistance,
of which serine deprivation could promote resistance to cisplatin in gastric cancer. As the key enzyme in the de novo
biosynthesis of serine, phosphoglycerate dehydrogenase (PHGDH) inhibition could also induce cisplatin resistance in
gastric cancer. This study aims to reveal the potential mechanisms of drug resistance induced by PHGDH inhibition via
exploring the global mRNA expression profiles.
Materials and Methods: In this experimental study, the viability and the apoptotic rate of gastric cancer cells
were evaluated by using Cell Counting Kit-8 (CCK-8) analysis and flow cytometric determination, respectively. The
identification of differentially expressed genes (DEGs) was tested by mRNA-sequencing (mRNA-Seq) analysis. The
confirmation of sequencing results was verified using real-time quantitative reverse transcription polymerase chain
reaction (RT-qPCR).
Results: The inhibition of PHGDH significantly increased the viability and decreased the apoptotic rate induced by cisplatin
in gastric cancer cells. mRNA-Seq analysis revealed that the combined treatment of NCT503 reduced the number of DEGs
induced by cisplatin. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment
Analysis (GSEA) showed that unfolded protein response, ECM receptor interaction and cell cycle signaling pathways were
modulated by NCT503 treatment. Hub genes were identified by using protein-protein interaction network modeling, of which E1A binding protein p300 (EP300) and heat shock protein family A (Hsp70) member 8 (HSPA8) act as the vital genes in cisplatin resistance induced by the inhibition of PHGDH.
Conclusion: These findings suggested that the inhibition of PHGDH promoted cisplatin resistance in gastric cancer
through various intercellular mechanisms. And appropriate serine supplementation or the modulation of EP300 and
HSPA8 may be of great help in overcoming cisplatin resistance in gastric cancer.

STUB1-SMYD2 Axis Regulates Drug Resistance in Glioma cells.

SET and MYND domain-containing protein 2 (SMYD2) is an important epigenetic regulator that methylates histone and non-histone proteins. The study aimed to investigate the oncogenic role of SMYD2 in gliomas and explore its degradation mechanism induced by cisplatin. Tumor tissue microarray of 441 patients with glioma was collected for SMYD2 immunohistochemical staining. Kaplan-Meier survival curves were constructed using the overall survival values. mRNA-sequencing analysis was performed for understanding the downstream mechanisms mediated by SMYD2. The half-inhibitory concentrations (IC50) of temozolomide and cisplatin in AZ505-treated and control cells were calculated. The potential E3 ubiquitin ligase of SMYD2 was predicted in UbiBrowser and confirmed by a knockdown test. The effect of SMYD2 and its E3 ligase on apoptosis and migration of glioma cells was determined via cell-function assays. High SMYD2 expression correlated with a high WHO stage (P = 0.004) and a low survival probability (P = 0.012). The inhibition of SMYD2 suppressed the process of epithelial to mesenchymal transition (EMT) by downregulating the expression of Collagen 1A1 (COL1A1). AZ505 treatment significantly increased the drug sensitivity of glioma cells. SMYD2 expression was markedly reduced by cisplatin treatment via STIP1 Homology And U-Box Containing Protein 1 (STUB1)-mediated degradation. The knockdown of STUB1 could partly reverse the cell function impairment induced by cisplatin. Our findings suggested that SMYD2 could be a potential drug target for the treatment of gliomas, and STUB1-mediated degradation of SMYD2 plays an important role in reversing chemotherapy resistance in patients with gliomas.

Specific metabolic response of patient-derived organoids to curcumin of colorectal cancer.

Organoid is a burgeoning model that have emerged in the past decade. Tumor organoids can simulate specific aspects of the 3D structure, cell type composition and function of real tumors to make up for the deficiencies of cell models and animal models. Curcumin has been found to be effective in suppressing various phases of colorectal cancer development. Nevertheless, there is no clear evidence that the results obtained on cultured cells or animal models can be translated in humans. Therefore, we constructed patient-derived organoids of colorectal cancer to show the curcumin responses of these organoids. Then, a MS-based non-targeted metabolomic strategy was to gain a system-level understanding of the mechanism of curcumin on colorectal cancer patient-derived organoids. Then non-targeted metabonomic analysis found that curcumin mainly regulated the phenylalanine, tyrosine and tryptophan biosynthesis, nicotinate and nicotinamide metabolism, purine metabolism in the organoids of colorectal cancer. Our research provided a reference for further revealing the role of curcumin in human-derived colorectal cancer-like solid tumors.

Role of Ubiquitin-Specific Peptidase 47 in Cancers and Other Diseases.

Deubiquitination is the reverse process of ubiquitination, which is catalyzed by deubiquitinase enzymes. More than 100 deubiquitinases have been identified. Ubiquitin-specific peptidase 47 (USP47), a member of the ubiquitin-specific protease family with high homology to USP7, is an active molecule with a wide range of functions and is closely associated with cancer and other diseases. However, no systematic summary exists regarding the functions of USP47. Here, we summarize the functions and expression regulation of USP47. USP47 is highly expressed in many tumors and is widely involved in tumor development, metastasis, drug resistance, epithelial-mesenchymal transition, and other processes. Targeted inhibition of USP47 can reverse malignant tumor behavior. USP47 also plays a role in inflammatory responses, myocardial infarction, and neuronal development. USP47 is involved in multiple levels of expression-regulating mechanisms, including transcriptional, post-transcriptional, and post-translational modifications. Development of targeted inhibitors against USP47 will provide a basis for studying the mechanisms of USP47 and developing therapeutic strategies for cancers and other diseases.

Serine Metabolism Regulates YAP Activity Through USP7 in Colon Cancer.

Metabolic reprogramming is a vital factor in the development of many types of cancer, including colon cancer. Serine metabolic reprogramming is a major feature of tumor metabolism. Yes-associated protein (YAP) participates in organ size control and tumorigenesis. However, the relationship between YAP and serine metabolism in colon cancer is unclear. In this study, RNA sequencing and metabolomics analyses indicated significant enrichment of the glycine, serine, and threonine metabolism pathways in serine starvation-resistant cells. Short-term serine deficiency inhibited YAP activation, whereas a prolonged response dephosphorylated YAP and promoted its activity. Mechanistically, USP7 increases YAP stability under increased serine conditions by regulating deubiquitination. Verteporfin (VP) effectively inhibited the proliferation of colon cancer cells and organoids and could even modulate serine metabolism by inhibiting USP7 expression. Clinically, YAP was significantly activated in colon tumor tissues and positively correlated with the expression of phosphoglycerate dehydrogenase (PHGDH) and USP7. Generally, our study uncovered the mechanism by which serine metabolism regulates YAP via USP7 and identified the crucial role of YAP in the regulation of cell proliferation and tumor growth; thus, VP may be a new treatment for colon cancer.

SYVN1-MTR4-MAT2A Signaling Axis Regulates Methionine Metabolism in Glioma Cells.

Methionine is one of the essential amino acids. How tumor cells adapt and adjust their signal transduction networks to avoid apoptosis in a methionine-restricted environment is worthy of further exploration. In this study, we investigated the molecular mechanism of glioma response to methionine restriction, providing a theoretical basis for new treatment strategies for glioma. METHODS: We constructed methionine-restriction-tolerant cells in order to study the response of glioma to a methionine-restricted environment. The transcriptome analysis of the tolerant cells showed significant changes in MAT2A. Western blotting, immunohistochemistry, quantitative real-time PCR, colony formation assays, and other experiments were used to verify the role of MAT2A in glioma genesis. In addition, the regulatory mechanism of MAT2A mRNA nuclear export was investigated by transfection, plasma nucleation separation, and co-immunoprecipitation. RESULTS: Under methionine restriction, glioma cells showed high expression of MAT2A, and an inhibitor of MAT2A reduced the proliferation of tumor cells. The expression of MAT2A was positively correlated with World Health Organization-grade glioma. High expression of MAT2A was related to increased transfer of its mRNA out of the nucleus. The expression of nuclear export regulatory molecule MTR4 could affect the export of MAT2A mRNA. In a methionine-restricted environment, ubiquitination of MTR4 was enhanced, and thus its protein level was reduced. The E3 ubiquitin ligase was verified to be SYVN1. CONCLUSION: In summary, methionine restriction leads to increased ubiquitination of MTR4, which promotes the transfer of MAT2A mRNA out of the nucleus and MAT2A protein expression. MAT2A promotes histone methylation, prompting cells to proliferate in a methionine-restricted environment.

Tanshinone IIA Improves Depression-like Behavior in Mice by Activating the ERK-CREB-BDNF Signaling Pathway.

Depression is a serious global affective disorder and one of the most common neurological diseases. Tanshinone IIA (TSA) is the mainly active constituent of Salvia miltiorrhiza and has diverse biological effects, including anti-inflammatory and antioxidant effects and significant neuroprotective effects against cerebral ischemia and Alzheimer's disease. However, whether TSA has an antidepressant effect remains unknown. The present study attempted to explore the antidepressant effects and the mechanism of TSA by examining the brain-derived neurotrophic factor (BDNF) expression in the hippocampus of depressive mice. The tail suspension test (TST) and forced swim test (FST) showed that TSA can significantly reduce the immobility time of depressed mice. Chronic administration of TSA increased p-ERK and p-CREB, BDNF proteins in mice hippocampus. We further explored the potential mechanism of TSA' antidepressant effect. TSA significantly increased the expression of p-ERK, p-CREB and BDNF proteins in dexamethasone-treated PC12 cells, and this enhancement was suppressed by pretreatment with the extracellular signal-regulated kinase (ERK) inhibitor SL327. Moreover, we observed that SL327 treatment markedly suppressed the increased levels of p-ERK, p-CREB and BDNF in mice hippocampus induced by TSA, preventing the antidepressant effects of TSA. Taken together, our results suggest that the antidepressant-like effects of TSA were mediated by ERK-CREB-BDNF pathway in mice hippocampus.

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.

DNA hydroxymethylation changes in response to spinal cord damage in a multiple sclerosis mouse model.

AIM: Roles of DNA 5-hydroxymethylcytosine (5hmC) in myelin repair were investigated in an experimental autoimmune encephalomyelitis (EAE) mouse model via its regulation on BDNF. METHODS: DNA 5hmC level and its limiting enzymes were detected in EAE mice. RESULTS: Global 5hmC modification, Tet1 and Tet2 significantly decreased in the spinal cord tissues of EAE mice. BDNF protein and mRNA decreased and were highly associated with BDNF 5hmC. Vitamin C, a Tet co-factor, increased global DNA 5hmC and reduced the neurological deficits at least by increasing BDNF 5hmC modification and protein levels. CONCLUSION: Tet protein-mediated 5hmC modifications represent a critical target involved in EAE-induced myelin damage. Targeting epigenetic modification may be a therapeutic strategy for multiple sclerosis.

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.

Application of GelGreen™ in Cesium Chloride Density Gradients for DNA-Stable Isotope Probing Experiments.

In this study, GelGreen™ was investigated as a replacement for SYBR® Safe to stain DNA in cesium chloride (CsCl) density gradients for DNA-stable isotope probing (SIP) experiments. Using environmental DNA, the usage of GelGreen™ was optimized for sensitivity compared to SYBR® Safe, its optimal concentration, detection limit for environmental DNA and its application in environmental DNA-SIP assay. Results showed that GelGreen™ was more sensitive than SYBR® Safe, while the optimal dosage (15X concentration) needed was approximately one-third of SYBR® Safe, suggesting that its sensitivity was three times more superior than SYBR® Safe. At these optimal parameters, the detection limit of GelGreen™-stained environmental DNA was as low as 0.2 µg, but the usage of 0.5 µg environmental DNA was recommended to produce a more consistent DNA band. In addition, a modified needle extraction procedure was developed to withdraw DNA effectively by fractionating CsCl density gradients into four or five fractions. The successful application of GelGreen™ staining with 13C-labeled DNA from enriched activated sludge suggests that this stain was an excellent alternative of SYBR® Safe in CsCl density gradients for DNA-SIP assays.