The impact of assisted reproductive technologies on ADHD: A systematic review and meta-analysis.

BACKGROUND: The escalating utilization of assisted reproductive technology (ART) in response to global infertility rates has spurred research into its complications. Short-term and long-term outcomes have been extensively studied, particularly the neurological concerns surrounding attention-deficit/hyperactivity disorder (ADHD) among ART-conceived children. This study aims investigate the association between ART and ADHD. METHODS: Medline, Embase, Scopus, and Web of Science databases were searched through April 4, 2023. Cohort, case-control, and cross-sectional studies were eligible for inclusion. primary summary measures included the unadjusted relative risk (RR) and adjusted hazard ratio (HR) with 95 % confidence intervals. Both fixed-effects and random-effects models were utilized for meta-analysis data pooling to determine the overall effect size. The onset of ADHD in children conceived through ART compared to those conceived naturally. RESULTS: The systematic search yielded 8 studies with 10,176,148 individuals included in the meta-analysis. The meta-analysis revealed a pooled RR of 0.93 (0.68-1.26) for cohort studies and a pooled RR of 0.97 (0.41-2.29) for cross-sectional studies, along with a pooled HR of 1.08 (1.03-1.13) for ADHD in the ART group compared to the non-ART group. CONCLUSION: While this study identifies some potential association between ART and ADHD, the limited effect size and inherent heterogeneity underscore the need for cautious interpretation.

Insight into the latitudinal gradient of biodiversity based on spatial variations in pelagic ciliate communities along the western Arctic Ocean.

The latitudinal dynamics of biodiversity has been the focus of global attention. This study is based on the latitude gradient of biodiversity in the spatial changes of pelagic ciliate communities in the western Arctic Ocean. The gradient pattern of pelagic ciliate communities across four latitudes were investigated from the water surface at 22 sampling station in the northern Bering Sea of the western Arctic Ocean and Chukchi Sea from August 5 to August 24, 2016. Based on multivariate analyses, the results showed that (1) the spatial patterns of pelagic ciliates represented a significant latitudinal gradient along the western Arctic Ocean; (2) the species number and abundance of pelagic ciliate communities declined from 64°N to 80°N; (3) variations in the horizontal distribution of ciliates were significantly correlated with changes in physicochemical variables, especially water temperature and Chl a; Thus it is suggested that the expected latitudinal decline of biodiversity was evident along the western Arctic Ocean.

Aerobic and anoxic utilization of organic matter for flexible nitrite supply in nutrient conversion pathways based on anaerobic ammonium oxidation: Microbial interactive mechanism.

This study investigated nutrient conversion pathways and corresponding interactive mechanisms in a mainstream partial-nitritation (PN)/anaerobic ammonium oxidation (anammox)/partial-denitrification-(PD)-enhanced biological phosphorus-removal (EBPR) (PN/A/PD-EBPR) process. A laboratory-scale sequencing batch reactor was operated for 301 days under different operational strategies. Mainstream PN/A/PD-EBPR was successfully operated with aerobic and anoxic utilization of organic matter. Aerobic utilization of organic matter was an effective strategy for conversion to denitrifying polyphosphate-accumulating organism-based phosphorus removal, referring to a biological reaction that outperformed nitrite-oxidizing bacteria. Aerobically adsorbed organic matter could be used as a carbon source for PD, which further enhanced nitrogen removal by PN/A. Ultimately, the interaction between complex nutrient conversion pathways served to achieve stable performance. High-throughput sequencing results elucidated the core microbe functioning in the mainstream PN/A/PD-EBPR process with respect to various nutrients. The outcomes of this study will be beneficial to those attempting to implement mainstream PN/A/PD-EBPR.

Transcriptomic Insights into Archaeal Nitrification in the Amundsen Sea Polynya, Antarctica.

Antarctic polynyas have the highest Southern Ocean summer primary productivity, and due to anthropogenic climate change, these areas have formed faster recently. Ammonia-oxidizing archaea (AOA) are among the most ubiquitous and abundant microorganisms in the ocean and play a primary role in the global nitrogen cycle. We utilized metagenomics and metatranscriptomics to gain insights into the physiology and metabolism of AOA in polar oceans, which are associated with ecosystem functioning. A polar-specific ecotype of AOA, from the "Candidatus Nitrosomarinus"-like group, was observed to be dominant in the Amundsen Sea Polynya (ASP), West Antarctica, during a succession of summer phytoplankton blooms. AOA had the highest transcriptional activity among prokaryotes during the bloom decline phase (DC). Metatranscriptomic analysis of key genes involved in ammonia oxidation, carbon fixation, transport, and cell division indicated that this polar AOA ecotype was actively involved in nitrification in the bloom DC in the ASP. This study revealed the physiological and metabolic traits of this key polar-type AOA in response to phytoplankton blooms in the ASP and provided insights into AOA functions in polar oceans.

Salinity-controlled distribution of prokaryotic communities in the Arctic sea-ice melt ponds.

The thawing of snow and sea ice produces distinctive melt ponds on the surface of the Arctic sea ice, which covers a significant portion of the surface sea ice during summer. Melt-pond salinity impacts heat transfer to the ice below and the melting rate. It is widely known that melt ponds play a significant role in heat fluxes, ice-albedo feedback, and sea-ice energy balance. However, not much attention has been given to the fact that melt ponds also serve as a unique microbial ecosystem where microbial production begins as soon as they are formed. Here, we investigated the role of melt pond salinity in controlling the diversity and distribution of prokaryotic communities using culture-dependent and -independent approaches. The 16 S rRNA gene amplicon based next generation sequencing analysis retrieved a total of 14 bacterial phyla, consisting of 146 genera, in addition to two archaeal phyla. Further, the culture-dependent approaches of the study allowed for the isolation and identification of twenty-four bacterial genera in pure culture. Flavobacterium, Candidatus_Aquiluna, SAR11 clade, Polaribacter, Glaciecola, and Nonlabens were the dominant genera observed in the amplicon analysis. Whereas Actimicrobium, Rhodoglobus, Flavobacterium, and Pseudomonas were dominated in the culturable fraction. Our results also demonstrated that salinity, chlorophyll a, and dissolved organic carbon were the significant environmental variables controlling the prokaryotic community distribution in melt ponds. A significant community shift was observed in melt ponds when the salinity changed with the progression of melting and deepening of ponds. Different communities were found to be dominant in melt ponds with different salinity ranges. It was also observed that melt pond prokaryotic communities significantly differed from the surface ocean microbial community. Our observations suggest that complex prokaryotic communities develop in melt ponds immediately after its formation using dissolved organic carbon generated through primary production in the oligotrophic water.

Atlantic-origin water extension into the Pacific Arctic induced an anomalous biogeochemical event.

The Arctic Ocean is facing dramatic environmental and ecosystem changes. In this context, an international multiship survey project was undertaken in 2020 to obtain current baseline data. During the survey, unusually low dissolved oxygen and acidified water were found in a high-seas fishable area of the western (Pacific-side) Arctic Ocean. Herein, we show that the Beaufort Gyre shrinks to the east of an ocean ridge and forms a front between the water within the gyre and the water from the eastern (Atlantic-side) Arctic. That phenomenon triggers a frontal northward flow along the ocean ridge. This flow likely transports the low oxygen and acidified water toward the high-seas fishable area; similar biogeochemical properties had previously been observed only on the shelf-slope north of the East Siberian Sea.

Divergent single cell transcriptome and epigenome alterations in ALS and FTD patients with C9orf72 mutation.

A repeat expansion in the C9orf72 (C9) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we investigate single nucleus transcriptomics (snRNA-seq) and epigenomics (snATAC-seq) in postmortem motor and frontal cortices from C9-ALS, C9-FTD, and control donors. C9-ALS donors present pervasive alterations of gene expression with concordant changes in chromatin accessibility and histone modifications. The greatest alterations occur in upper and deep layer excitatory neurons, as well as in astrocytes. In neurons, the changes imply an increase in proteostasis, metabolism, and protein expression pathways, alongside a decrease in neuronal function. In astrocytes, the alterations suggest activation and structural remodeling. Conversely, C9-FTD donors have fewer high-quality neuronal nuclei in the frontal cortex and numerous gene expression changes in glial cells. These findings highlight a context-dependent molecular disruption in C9-ALS and C9-FTD, indicating unique effects across cell types, brain regions, and diseases.

Preventing Risks of Infections and Medication Errors in IV therapy (PRIME): a patient safety initiative.

BACKGROUND: Two major avoidable reasons for adverse events in hospital are medication errors and intravenous therapy-induced infections or complications. Training for clinical staff and compliance to patient safety principles could address these. METHODS: Joint Commission International (JCI) consultants created a standardised, 6-month training programme for clinical staff in hospitals. Twenty-one tertiary care hospitals from across south-east Asia took part. JCI trained the clinical consultants, who trained hospital safety champions, who trained nursing staff. Compliance and knowledge were assessed, and monthly audits were conducted. RESULTS: There was an overall increase of 29% in compliance with parameters around medication preparation and vascular access device management. CONCLUSION: The programme improved safe practice around preparing medications management and managing vascular access devices. The approach could be employed as a continuous quality improvement initiative for the prevention of medication errors and infusion-associated complications.

RNA outperforms DNA-based metabarcoding in assessing the diversity and response of microeukaryotes to environmental variables in the Arctic Ocean.

The Arctic Ocean (AO) has a harsh environment characterized by low temperatures, extensive ice coverage, and periodic freezing and melting of sea ice, which has provided diverse habitats for microorganisms. Prior studies primarily focused on microeukaryote communities in the upper water or sea ice based on environmental DNA, leaving the composition of active microeukaryotes in the diverse AO environments largely unknown. This study provided a vertical assessment of microeukaryote communities in the AO from snow and ice to sea water at a depth of 1670 m using high-throughput sequencing of co-extracted DNA and RNA. RNA extracts depicted microeukaryote community structure and intergroup correlations more accurately and responded more sensitively to environmental conditions than those derived from DNA. Using RNA:DNA ratios as a proxy for relative activity of major taxonomic groups, the metabolic activities of major microeukaryote groups were determined along depth. Analysis of co-occurrence networks showed that parasitism between Syndiniales and dinoflagellates/ciliates in the deep ocean may be significant. This study increased our knowledge of the diversity of active microeukaryote communities and highlighted the importance of using RNA-based sequencing over DNA-based sequencing to examine the relationship between microeukaryote assemblages and the responses of microeukaryotes to environmental variables in the AO.

Odor reduction using hydrogen sulfide-removing bacteria in sludge filtration systems: Ferrous-oxidizing bacteria and sulfur-oxidizing bacteria.

The preconditioning of digested sludge in sludge filtration systems produces hydrogen sulfide (H2S), a major odor-causing source. This study evaluated the effects of adding H2S-removing bacteria to sludge-filtration systems. Ferrous-oxidizing bacteria (FOB) and sulfur-oxidizing bacteria (SOB) were mass-cultivated in a hybrid bioreactor equipped with an internal circulation system. In this bioreactor, FOB and SOB effectively removed >99% of H2S; however, the acidic conditions created by adding a coagulant during digested sludge preconditioning were more favorable for FOB than for SOB. In batch tests, SOB and FOB removed 94 ± 1.1% and 99 ± 0.1% of H2S, respectively; therefore, digested sludge preconditioning proved more suitable for FOB activity than SOB activity. The results revealed that the optimal FOB addition ratio was 0.2%, validated using a pilot filtration system. Moreover, the 57.5 ± 2.9 ppm H2S generated in the sludge preconditioning step was reduced to 0.01 ± 0.01 ppm after adding 0.2% FOB. Therefore, the results of this study will be useful because they provide a process for biologically removing odor-causing sources without affecting the dewatering efficiency of the filtration system.

Elucidating prioritized factor for mainstream partial nitritation between C/N ratio and dissolved oxygen: Response surface methodology and microbial community shifts.

Recently, C/N ratio is suggested as a promising control factor with dissolved oxygen (DO) achieving mainstream partial nitritation (PN); however, their combined effects on mainstream PN are still limited. This study evaluated the mainstream PN with respect to the combined factors, and investigated the prioritized factor affecting the community of aerobic functional microbes competing with NOB. Response surface methodology was performed to assess the combined effects of C/N ratio and DO on the activity of functional microbes. Aerobic heterotrophic bacteria (AHB) played the greatest role in oxygen competition among functional microbes, which resulted in relative inhibition of nitrite-oxidizing bacteria (NOB). The combination of high C/N ratio and low DO had a positive role in the relative inhibition of NOB. In bioreactor operation, the PN was successfully achieved at ≥ 1.5 of C/N ratio for 0.5-2.0 mg/L DO conditions. Interestingly, aerobic functional microbes outcompeting NOB were shifted with C/N ratio rather than DO, suggesting C/N ratio is more prioritized factor achieving mainstream PN. These findings will provide insights into how combined aerobic conditions contribute to achieve mainstream PN.

Water Mass Controlled Vertical Stratification of Bacterial and Archaeal Communities in the Western Arctic Ocean During Summer Sea-Ice Melting.

The environmental variations and their interactions with the biosphere are vital in the Arctic Ocean during the summer sea-ice melting period in the current scenario of climate change. Hence, we analysed the vertical distribution of bacterial and archaeal communities in the western Arctic Ocean from sea surface melt-ponds to deep water up to a 3040 m depth. The distribution of microbial communities showed a clear stratification with significant differences among different water depths, and the water masses in the Arctic Ocean - surface mixed layer, Atlantic water mass and deep Arctic water - appeared as a major factor explaining their distribution in the water column. A total of 34 bacterial phyla were detected in the seawater and 10 bacterial phyla in melt-ponds. Proteobacteria was the dominant phyla in the seawater irrespective of depth, whereas Bacteroidota was the dominant phyla in the melt-ponds. A fast expectation-maximization microbial source tracking analysis revealed that only limited dispersion of the bacterial community was possible across the stratified water column. The surface water mass contributed 21% of the microbial community to the deep chlorophyll maximum (DCM), while the DCM waters contributed only 3% of the microbial communities to the deeper water masses. Atlantic water mass contributed 37% to the microbial community of the deep Arctic water. Oligotrophic heterotrophic bacteria were dominant in the melt-ponds and surface waters, whereas chemoautotrophic and mixotrophic bacterial and archaeal communities were abundant in deeper waters. Chlorophyll and ammonium were the major environmental factors that determined the surface microbial communities, whereas inorganic nutrient concentrations controlled the deep-water communities.

Ecological Importance of Viral Lysis as a Loss Factor of Phytoplankton in the Amundsen Sea.

Whether phytoplankton mortality is caused by grazing or viral lysis has important implications for phytoplankton dynamics and biogeochemical cycling. The ecological relevance of viral lysis for Antarctic phytoplankton is still under-studied. The Amundsen Sea is highly productive in spring and summer, especially in the Amundsen Sea Polynya (ASP), and very sensitive to global warming-induced ice-melt. This study reports on the importance of the viral lysis, compared to grazing, of pico- and nanophytoplankton, using the modified dilution method (based on apparent growth rates) in combination with flow cytometry and size fractionation. Considerable viral lysis was shown for all phytoplankton populations, independent of sampling location and cell size. In contrast, the average grazing rate was 116% higher for the larger nanophytoplankton, and grazing was also higher in the ASP (0.45 d-1 vs. 0.30 d-1 outside). Despite average specific viral lysis rates being lower than grazing rates (0.17 d-1 vs. 0.29 d-1), the average amount of phytoplankton carbon lost was similar (0.6 µg C L-1 d-1 each). The viral lysis of the larger-sized phytoplankton populations (including diatoms) and the high lysis rates of the abundant P. antarctica contributed substantially to the carbon lost. Our results demonstrate that viral lysis is a principal loss factor to consider for Southern Ocean phytoplankton communities and ecosystem production.

Factors affecting the subsurface aragonite undersaturation layer in the Pacific Arctic region.

This study evaluated interannual variation in the subsurface aragonite undersaturation zone (ΩAr<1 layer) in the Pacific Arctic Ocean, using data from the 2016-2019 period. The upper boundary (DEPΩ<1UB) of the ΩAr<1 layer generally formed at a depth where the contribution of corrosive Pacific water was approximately 98 %. The intensity of the Beaufort Gyre associated with freshwater accumulation mainly determined interannual variation in DEPΩ<1UB, but the direction of its effect was opposite west and east of ~166°W. The lower boundary (DEPΩ<1LB) of the ΩAr<1 layer was generally found at a depth range where equal contributions of Pacific and Atlantic water were expected. An Atlantic-origin cold saline water intrusion event in 2017 caused by an anomalous atmospheric circulation pattern significantly lifted the DEPΩ<1LB, thus the thickness of the ΩAr<1 layer decreased.

Effect of aerobic microbes' competition for oxygen on nitrogen removal in mainstream nitritation-anammox systems.

The effects of C/N ratio in mainstream partial nitritation (PN)-anaerobic ammonia oxidation (ANAMMOX) considering competitive relationship of aerobic microbes competing for oxygen were investigated. Thy system was operated for 501 d with various C/N ratio. Competitive growth of aerobic heterotrophic bacteria (AHB) at ≥ 1 of C/N ratio acted effectively on the selective inhibition of nitrite-oxidizing bacteria (NOB) while contributing to stable PN-A. In-depth kinetic analysis indicated oxygen affinity of aerobic microbes was in the order of AHB > ammonia-oxidizing bacteria (AOB) > NOB. In addition, potential of denitritation by AHB could contributed to improving nitrogen removal up to 87.5 ± 4.3%. AHB was comparatively clustered into two groups with a C/N ratio of 1. Nitrosomonas sp. PY1 became predominant while Nitrospira spp. were the major NOB. The potential of AHB in establishing selective inhibition of NOB was identified, which could be a novel approach to stabilze the mainstream PN-A.

Women's attitudes toward certification logos, labels, and advertisements for organic disposable sanitary pads: results from a multi-city cross-sectional survey.

BACKGROUND: This cross-sectional study evaluated women's attitudes toward the certification logos, labels, and advertisements for organic disposable sanitary pads (OSPs) and investigated what could be the main reason for them. Additionally, the present study examined whether a relationship could be found between these attitudes and OSPs purchasing behavior. METHODS: This study was conducted using a self-reported online survey of Korean adult women who have purchased OSPs. The study questionnaire had four sections, covering (1) characteristics of OSP purchasing behavior, (2) attitudes toward OSP certification logos, labels, and advertisements, (3) demand on government and companies for proper management, and (4) respondent's sociodemographic information. The Cronbach's alpha value of the questionnaire was 0.857. RESULTS: A total of 500 respondents completed the questionnaire. Overall, high reliability was found for the certification logos (3.73 ± 0.61), labels on the product packaging (3.71 ± 0.63), and advertisements of OSPs (3.41 ± 0.62). Respondents indicated that these had fairly positive effects on their decision-making regarding product reliability, product image, and their own purchasing behavior. The aspects most frequently affected from the informants were safety to human health. All attitudes toward OSP certification logos, labels, and advertisements that were evaluated in this study became more positive in the direction from non-buyers to occasional buyers and to habitual buyers (all P < 0.05). The most significant demand from the respondents for OSP companies and the government was to clearly indicate hazardous ingredients on the OSP packaging (42.0%) and to strengthen the sanctions for false advertising (37.8%), respectively. CONCLUSIONS: The results of this study clearly indicate the importance of using certification logos, labels, and advertisements in the OSP market. These results can be utilized by OSP companies to improve the effectiveness of their marketing strategies or by policy makers and certifying bodies to manage the informants properly in the OSP market.

Evaluation of operating parameters affecting the two-stage nitritatin/anammox process in mainstream flows: From lab-scale to pilot-scale.

Operating parameters for the two-stage nitritation/anammox (PN/A) process in mainstream flows from lab-scale to pilot-scale were investigated. In the batch experiment, the specific activities of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) were fitted with a Monod model, and the oxygen affinity of NOB was higher than that of AOB. Nevertheless, dissolved oxygen concentrations higher than 2.0 mgO2/L were effectively applied for selective inhibition of NOB. In addition, organic matter was evaluated as the operating parameter that inhibited the activity of NOB more selectively than AOB under mainstream conditions. The operating parameters determined in the batch experiment were verified in a continuous experiment, and it was shown that the shortening of hydraulic retention time with increasing aeration time in the sequencing batch reactor cycle has the potential to selectively enhance the activity of AOB over NOB. The effect on the C/N ratio and temperature in a lab-scale anammox reactor was investigated. The denitrification with the addition of organic matter contributed to the improvement of nitrogen removal efficiency, but a C/N ratio of more than 1.0 severely deteriorated the sludge settleability. The operating factors for the mainstream PN/A determined in the lab scale were applied to the pilot plant. As a result, a nitrogen removal efficiency of 79 ± 11% was stably achieved with effluent total nitrogen of 2.6 ± 1.5 mg/L. However, unpredictable fluctuations in environmental factors revealed that residual ammonium was another critical factor affecting mainstream PN/A.

Activation of N-acyl-homoserine lactone-mediated quorum sensing system improves long-term preservation of anammox microorganisms by vacuum lyophilization.

The long-term preservation of anaerobic ammonium oxidation (anammox) microorganisms via vacuum lyophilization process would help commercialize the technique. In this study, vacuum lyophilization was evaluated for the cost-effective long-term preservation of such microorganisms. Skim milk was found to be the most effective cryoprotectant for maintaining the physiological properties (heme c, EPS, and the PN/PS ratio) of anammox microorganisms. Conversely, the vacuum lyophilization technique was shown to cause serious damage to the quorum sensing (QS) system of anammox, so that anammox activity was not adequately recovered afterwards. To overcome this limitation, activation of the AHL-mediated QS system were applied to the vacuum lyophilization process. Endogenous (i.e., fresh anammox sludge of 10%) and exogenous (i.e., C6-HSL of 60 mg/L) QS autoinducers significantly increased anammox activity to 88.2 ± 12.2 and 130.0 ± 12.2 mgTN/gVSS/d, respectively, after 56 d of reactivation. In addition, nitrogen removal potentials were estimated to be 123.5 and 87.5 gTN/m3/d, respectively. The effect of the exogenous QS autoinducer on anammox reactivation was reconfirmed through the comparison experiment. The results of this study will be greatly significant to this field since they improve the feasibility of the once-underestimated vacuum lyophilization technique.

Spatial dynamics of active microeukaryotes along a latitudinal gradient: Diversity, assembly process, and co-occurrence relationships.

Recent global warming is profoundly and increasingly influencing the Arctic ecosystem. Understanding how microeukaryote communities respond to changes in the Arctic Ocean is crucial for understanding their roles in the biogeochemical cycles of nutrients and elements. Between July 22 and August 19, 2016, during cruise ARA07, seawater samples were collected along a latitudinal transect extending from the East Sea of Korea to the central Arctic Ocean. Environmental RNA was extracted and the V4 hypervariable regions of the reverse transcribed SSU rRNA were amplified. The sequences generated by high throughput sequencing were clustered into zero-radius OTUs (ZOTUs), and the taxonomic identities of each ZOTU were assigned using SINTAX against the PR2 database. Thus, the diversity, community composition, and co-occurrence networks of size fractionated microeukaryotes were revealed. The present study found: 1) the alpha diversity of pico- and nano-sized microeukaryotes showed a latitudinal diversity gradient; 2) three distinct communities were identified, i.e., the Leg-A, Leg-B surface, and Leg-B subsurface chlorophyll a maximum (SCM) groups; 3) distinct network structure and composition were found in the three groups; and 4) water temperature was identified as the primary factor driving both the alpha and beta diversities of microeukaryotes. This study conducted a comprehensive and systematic survey of active microeukaryotes along a latitudinal gradient, elucidated the diversity, community composition, co-occurrence relationships, and community assembly processes among major microeukaryote assemblages, and will help shed more light on our understanding of the responses of microeukaryote communities to the changing Arctic Ocean.

Evaluation of long-term preservation and reactivation efficiency of anaerobic ammonium oxidation (anammox) microorganisms based on activation energy.

The preservation efficiency of mainstream (M-ANA) and sidestream anaerobic ammonium oxidation (anammox) (S-ANA) were evaluated based on their activation energy (Ea). The Ea of M-ANA cultivated under low nitrogen loads was lower than that of S-ANA, which greatly contributed to enhancing the viability of anammox during preservation at 4 °C. After preservation for 140 d, the decay rate (bAN) of M-ANA ranged from 0.0012 to 0.0013/d; the bAN of S-ANA was 0.0036-0.0041/d. The addition of hydrazine, which requires minimal energy to activate anammox metabolism, is highly beneficial for the viability of microorganisms. The low Ea of anammox contributes to efficient reactivation with rapid reactivation of heme c, and the addition of hydrazine makes the process more beneficial. Although the specific nitrogen removal rate of the M-SNA seed sludge was much lower than that of S-ANA, the rate of M-ANA became higher after 48 days of reactivation.