Composting reduces the risks of resistome in beef cattle manure at the transcriptional level.

Transcriptomic evidence is needed to determine whether composting is more effective than conventional stockpiling in mitigating the risk of resistome in livestock manure. The objective of this study is to compare composting and stockpiling for their effectiveness in reducing the risk of antibiotic resistance in beef cattle manure. Samples collected from the center and the surface of full-size manure stockpiling and composting piles were subject to metagenomic and metatranscriptomic analyses. While the distinctions in resistome between stockpiled and composted manure were not evident at the DNA level, the advantages of composting over stockpiling were evident at the transcriptomic level in terms of the abundance of antibiotic resistance genes (ARGs), the number of ARG subtypes, and the prevalence of high-risk ARGs (i.e., mobile ARGs associated with zoonotic pathogens). DNA and transcript contigs show that the pathogen hosts of high-risk ARGs included Escherichia coli O157:H7 and O25b:H4, Klebsiella pneumoniae, and Salmonella enterica. Although the average daily temperatures for the entire composting pile exceeded 55°C throughout the field study, more ARG and ARG transcripts were removed at the center of the composting pile than at the surface. This work demonstrates the advantage of composting over stockpiling in reducing ARG risk in active populations in beef cattle manure.IMPORTANCEProper treatment of manure before land application is essential to mitigate the spread of antibiotic resistance in the environment. Stockpiling and composting are two commonly used methods for manure treatment. However, the effectiveness of composting in reducing antibiotic resistance in manure has been debated. This work compared the ability of these two methods to reduce the risk of antibiotic resistance in beef cattle manure. Our results demonstrate that composting reduced more high-risk resistance genes at the transcriptomic level in cattle manure than conventional stockpiling. This finding not only underscores the effectiveness of composting in reducing antibiotic resistance in manure but also highlights the importance of employing RNA analyses alongside DNA analyses.

Quorum sensing responses of r-/K-strategists Nitrospira in continuous flow and sequencing batch nitrifying biofilm reactors.

A better understanding of r-/K-strategists nitrifiers will help to balance the design and operation of bioprocesses for efficient pollution removal from wastewater. The objectives of study were to investigate the nitrite oxidation biokinetics, biofilm property, microbial community and quorum sensing (QS) of nitrifying biofilm in a continuously flow reactor (CFR) and a sequencing batch reactor (SBR). Results showed that nitrite-oxidizing bacteria were estimated to have a nitrite half saturation constant of 76.23 and 224.73 µM in CFR and SBR, respectively. High-throughput and metagenomic sequencing results showed that Nitrospira and Candidatus Nitrospira defluvii were the dominated nitrite-oxidizing taxa performing nitrite oxidation in both reactors. Nitrifying biofilm developed in CFR and SBR showed obviously different properties. Biofilm in SBR had an obviously higher ratio of polysaccharide and protein in extracellular polymeric substances, and higher thickness than in CFR. Metagenomics and chemical analysis revealed various types of acyl-homoserine lactone (AHL) circuit genes (e.g., luxI, lasI, hdtS) and four types of AHL signaling substances (e.g., C6-HSL, C8-HSL, C10-HSL and 3-oxo-C10-HSL) in nitrifying biofilm. The concentrations of these AHLs in biomass and water phases were obviously higher in SBR than that in CFR. Together, AHLs-based QS might affect the formation of nitrifying biofilm and thus contribute to the different biokinetics of Nitrospira in CFR and SBR. Our insights may reveal the molecular mechanism of Nitrospira for different biokinetics, and indicate the AHL association with Nitrospira adaptation to various conditions.

Aerobic nitrous oxide emission in anoxic/aerobic and intermittent aeration sequencing batch reactors.

This study characterized nitrogen removal and nitrous oxide (N2O) emissions from lab-scale anoxic/aerobic sequencing batch reactor (AOSBR) and intermittent aeration sequencing batch reactor (IASBR), respectively, for treating synthetic municipal wastewater. The N2O emission was evaluated in a simulated cycle, and batch conditions of aerobic nitrification, simultaneous nitrification and denitrification (SND), and aerobic denitrification. The results show that nitrogen removal was enhanced in IASBR compared to AOSBR, with 94.2% and 67.9% of total inorganic nitrogen removal efficiency in IASBR and AOSBR, respectively. In the simulated cycle, the emission factors (of oxidized ammonium) were 4.9% and 0.6% in AOSBR and IASBR, respectively. Under batch conditions, the N2O emission factors during SND were obviously higher than that during aerobic nitrification and denitrification. The N2O emission factors during SND ranging 0.68-11.68% in AOSBR and 1.25-5.13% in IASBR. Furthermore, N2O emission under batch conditions was affected by the aeration ratios. Moderate and high aeration ratios used in this study stimulated the N2O emission from SND. The N2O emission was enhanced with the nitrite accumulation during aerobic nitrification when the nitrite-oxidizing bacteria was inhibited by a chemical inhibitor. Aerobic denitrification via nitrite could be the main pathway of N2O generation from SND processes. The findings from our study can help further understand N2O emission mechanisms and guide the optimization of the current wastewater treatment process for minimizing N2O emission.

Fate of antibiotic resistance genes and class 1 integrons during sludge treatment using pilot-scale anaerobic digestion with thermal hydrolysis pretreatment.

This work evaluated the fate of antibiotic resistance genes (ARGs) and class 1 integron gene in sewage sludge before and after pilot-scale thermal hydrolysis pretreatment (THP) and subsequent mesophilic anaerobic digestion (AD) treatment. Variables investigated include THP temperatures, feedstock types, and AD solids retention times. Real-time polymerase chain reaction was performed to quantify the ARGs in feedstocks, THP and AD effluent. Results show that THP significantly (t test, p < 0.05) reduced the absolute abundances of most ARGs, with the reduction ranging from 0.03 to 3.09 log units. Rebound effects of ARGs in the subsequent AD were observed and were relevant with tested variables; shorter solids retention time (10 days) and higher THP temperature (165 ℃) can significantly reduce ARGs in AD effluent. These findings provide references about the effects of the THP and AD on the control of ARG spread from sewage sludge to environments.

Unblocking the rate-limiting step of the municipal sludge anaerobic digestion.

Anaerobic digestion stabilizes municipal sludge through total solids reduction and biogas production. It is generally accepted that hydrolysis accounts for the rate-limiting step of municipal sludge anaerobic digestion, impacting the overall rates of solids reduction and methane production. Technically, the sludge hydrolysis rate can be enhanced by the application of thermal hydrolysis pretreatment (THP) and is also affected by the total solids concentration, temperature, and solids retention time used in the anaerobic digestion. This study systematically analyzed and compared ways to take these four factors into the consideration of modern anaerobic digestion system for achieving the maximum solid reduction. Results showed that thermophilic anaerobic digestion was superior to mesophilic anaerobic digestion in terms of solids reduction but vice versa in terms of the methane production when integrated with THP. This difference has to do with the intermediate product accumulation and inhibition when hydrolysis outpaced methanogenesis in THP-enhanced thermophilic anaerobic digestion, which can be mitigated by adjusting the solids retention time. PRACTITIONER POINTS: THP followed by TAD offers the greatest solids reduction rate. THP followed by MAD offered the greatest methane production rate. FAN inhibition appears to be an ultimate limiting factor constraining the methane production rate. In situ ammonia removal technique should be developed to further unblock the rate-limiting step.

Effects of Nutrient Level and Growth Rate on the Conjugation Process That Transfers Mobile Antibiotic Resistance Genes in Continuous Cultures.

Bacteria in the effluent of wastewater treatment plants (WWTPs) can transfer antibiotic resistance genes (ARGs) to the bacteria in receiving water through conjugation; however, there is a lack of quantitative assessment of this phenomenon in continuous cultures. Our objective was to determine the effects of background nutrient levels in river water column and growth rates of bacteria on the conjugation frequency of ARGs from effluent bacteria to river bacteria, as well as on the resulting resistance level (i.e., MICs) of the river bacteria. Chemostats were employed to simulate the discharge points of WWTPs into rivers, where effluent bacteria (donor cells) meet river bacteria (recipient cells). Both donor and recipient cells were Escherichia coli cells, and the donor cells were constructed by filter mating with bacteria in the effluent of a local WWTP. Results showed that higher bacterial growth rate (0.45 h-1 versus 0.15 h-1) led to higher conjugation frequencies (10-4 versus 10-6 transconjugant per recipient). The nutrient level also significantly affected the conjugation frequency, albeit to a lesser extent than the growth rate. The MIC against tetracycline increased from 2 mg/L in the recipient to 64 to 128 mg/L in transconjugants. In comparison, the MIC only increased to as high as 8 mg/L in mutants. Whole-genome sequencing showed that the tet-containing plasmid in both the donor and the transconjugant cells also occur in other fecal bacterial genera. The quantitative information obtained from this study can inform hazard identification related to the proliferation of wastewater-associated ARGs in surface water. IMPORTANCE WWTPs have been regarded as an important hot spot of ARGs. The discharge point of WWTP effluent, where ARGs may be horizontally transferred from bacteria of treated wastewater to bacteria of receiving water, is an important interface between the human-dominated ecosystem and the natural environment. The use of batch cultures in previous studies cannot adequately simulate the nutrient conditions and growth rates in receiving water. In this study, chemostats were employed to simulate the continuous growth of bacteria in receiving water. Furthermore, the experimental setup allowed for separate investigations on the effects of nutrient levels (i.e., simulating background nutrients in river water) and bacterial growth rates on conjugation frequencies and resulting resistance levels. The study generates statistically sound ecological data that can be used to estimate the risk of wastewater-originated ARGs as part of the One Health framework.

Pivotal role of municipal wastewater resource recovery facilities in urban agriculture: A review.

Urban agriculture provides a promising, comprehensive solution to water, energy, and food scarcity challenges resulting from the population growth, urbanization, and the accelerating effects of anthropogenic climate change. Their close access to consumers, profitable business models, and important roles in educational, social, and physical entertainment benefit both developing and developed nations. In this sense, Urban Water Resource Reclamation Facilities (WRRFs) can play a pivotal role in the sustainable implementation of urban agriculture. Reclaimed water as a recovered resource has less supply variability and in certain cases can be of higher quality than other water sources used in agriculture. Another recovered resource, namely, biosolids, as byproduct from wastewater treatment can be put to beneficial use as fertilizers, soil amendments, and construction material additives. The renewable electricity, heat, CO2 , and bioplastics produced from WRRFs can also serve as essential resources in support of urban agriculture operation with enhanced sustainability. In short, this review exhibits a holistic picture of the state-of-the-art of urban agriculture in which WRRFs can potentially play a pivotal role. PRACTITIONER POINTS: Reclaimed water can be of higher quality than other sources used in urban agriculture. Biosolids can be put to beneficial use as fertilizers, soil amendments, and construction material additives. The renewable electricity, heat, CO2 , and bioplastics produced can also serve as essential resources in support of urban agriculture.

The r/K selection theory and its application in biological wastewater treatment processes.

Understanding the characteristics of functional organisms is the key to managing and updating biological processes for wastewater treatment. This review, for the first time, systematically characterized two typical types of strategists in wastewater treatment ecosystems via the r/K selection theory and provided novel strategies for selectively enriching microbial community. Functional organisms involved in nitrification (e.g., Nitrosomonas and Nitrosococcus), anammox (Candidatus Brocadia), and methanogenesis (Methanosarcinaceae) are identified as r-strategists with fast growth capacities and low substrate affinities. These r-strategists can achieve high pollutant removal loading rates. On the other hand, other organisms such as Nitrosospira spp., Candidatus Kuenenia, and Methanosaetaceae, are characterized as K-strategists with slow growth rates but high substrate affinities, which can decrease the pollutant concentration to low levels. More importantly, K-strategists may play crucial roles in the biodegradation of recalcitrant organic pollutants. The food-to-microorganism ratio, mass transfer, cell size, and biomass morphology are the key factors determining the selection of r-/K-strategists. These factors can be related with operating parameters (e.g., solids and hydraulic retention time), biomass morphology (biofilm or granules), and operating modes (continuous-flow or sequencing batch), etc., to achieve the efficient acclimation of targeted r-/K-strategists. For practical applications, the concept of substrate flux was put forward to further benefit the selective enrichment of r-/K-strategists, fulfilling effective management and improvement of engineered pollution control bioprocesses. Finally, the future perspectives regarding the development of the r/K selection theory in wastewater treatment processes were discussed.

Predicting antibiotic resistance gene abundance in activated sludge using shotgun metagenomics and machine learning.

While the microbiome of activated sludge (AS) in wastewater treatment plants (WWTPs) plays a vital role in shaping the resistome, identifying the potential bacterial hosts of antibiotic resistance genes (ARGs) in WWTPs remains challenging. The objective of this study is to explore the feasibility of using a machine learning approach, random forests (RF's), to identify the strength of associations between ARGs and bacterial taxa in metagenomic datasets from the activated sludge of WWTPs. Our results show that the abundance of select ARGs can be predicted by RF's using abundant genera (Candidatus Accumulibacter, Dechloromonas, Pesudomonas, and Thauera, etc.), (opportunistic) pathogens and indicators (Bacteroides, Clostridium, and Streptococcus, etc.), and nitrifiers (Nitrosomonas and Nitrospira, etc.) as explanatory variables. The correlations between predicted and observed abundance of ARGs (erm(B), tet(O), tet(Q), etc.) ranged from medium (0.400 < R2 < 0.600) to strong (R2 > 0.600) when validated on testing datasets. Compared to those belonging to the other two groups, individual genera in the group of (opportunistic) pathogens and indicator bacteria had more positive functional relationships with select ARGs, suggesting genera in this group (e.g., Bacteroides, Clostridium, and Streptococcus) may be hosts of select ARGs. Furthermore, RF's with (opportunistic) pathogens and indicators as explanatory variables were used to predict the abundance of select ARGs in a full-scale WWTP successfully. Machine learning approaches such as RF's can potentially identify bacterial hosts of ARGs and reveal possible functional relationships between the ARGs and microbial community in the AS of WWTPs.

Transmission Routes of the Microbiome and Resistome from Manure to Soil and Lettuce.

The land application of animal manure can introduce manure microbiome and resistome to croplands where food crops are grown. The objective of this study was to characterize the microbiome and resistome on and in the leaves of lettuce grown in manured soil and identify the main transmission routes of microbes and antibiotic resistance genes (ARGs) from soil to the episphere and endosphere of lettuce. Shotgun metagenomic results show that manure application significantly altered the composition of the microbiome and resistome of surface soil. SourceTracker analyses indicate that manure and original soil were the main source of the microbiome and resistome of the surface soil and rhizosphere soil, respectively. Manure application altered the microbiome and resistome in the episphere of lettuce (ADONIS p < 0.05), and surface soil accounted for ∼81% of the microbes and ∼62% of the ARGs in episphere. Manure application had limited impacts on the microbiome and resistome in the endosphere (ADONIS p > 0.05). Our results show that manure-borne microbes and ARGs reached the episphere primarily through surface soil and some epiphytic microbes and ARGs further entered the endosphere. Our findings can inform the development of pre- and postharvest practices to minimize the transmission of manure-borne resistome from food crops to consumers.

Potential microbial functions and quorum sensing systems in partial nitritation and anammox processes.

Diverse microbial communities coexist in the partial nitritation-anaerobic ammonium oxidation (PNA) process, in which nitrogen metabolism and information exchange are two important microbial interactions. In the PNA process, the existence of diverse microorganisms including nitrifiers, anammox bacteria, and heterotrophs makes it challenging to achieve a balanced relationship between anaerobic ammonium oxidation bacteria and ammonia oxidizing bacteria. In this study, potential microbial functions in nitrogen conversion and acyl-homoserine lactones (AHLs)-based quorum sensing (QS) in PNA processes were examined. Candidatus_Kuenenia and Nitrosomonas were the key functional bacteria responsible for PNA, while Nitrospira was detected as the dominant nitrite oxidizing bacteria (NOB). Heterotrophs containing nxr might play a similar function to NOB. The AHLs-QS system was an important microbial communication pathway in PNA systems. N-octanoyl-L-homoserine lactone, N-decanoyl homoserine lactone, and N-dodecanoyl homoserine lactone were the main AHLs, which might be synthesized by nitrogen converting microorganisms and heterotrophs. However, only heterotrophs had the potential to sense and degrade AHLs, such as Saccharophagus (sensing) and Leptospira (degradation). These results provide comprehensive information about the possible microbial functions and interactions in the PNA system and clues for system optimization from a microbial perspective. PRACTITIONER POINTS: ●Potential functions of anammox bacteria, nitrifiers, and heterotrophs were revealed. ●Diverse nitrogen conversion and AHLs-quorum sensing related genes were detected. ●Anammox bacteria and AOB played important roles in the AHLs synthesis process. ●Heterotrophs could sense and degrade AHLs during information exchange.

Different moxibustion therapies for urinary retention after anorectal surgery: A protocol for systematic review and network meta-analysis.

BACKGROUND: Postoperative urinary retention is a disease that seriously affects human daily work and life, and greatly reduces people's quality of life and affects human health all over the world. Now, many studies have shown that moxibustion has a significant effect on postoperative urinary retention. In this study, network meta-analysis was used to analyze and compare the clinical efficacy and difference of different moxibustion treatments on postoperative urinary retention. METHODS: Only randomized controlled trials (RCTs) will be included and all patients were diagnosed as postoperative urinary retention. Computer search Chinese databases: CNKI, Wanfang (WANFANG), VIP (VIP), Chinese Biomedical Literature Database (SinoMed), English database search PubMed, Cochrane library, Web of Science. The search period limit is from the time the date of database establishment to November 17, 2020. To avoid omissions, we will manually search for relevant reference materials and conference papers. The risk of bias in the final included studies will be assessed according to the guidelines of the Cochrane System Intervention Review Manual. All data analysis will be conducted by Revman5.3, Gemtc 0.14.3, and Stata 14.2. RESULTS: The effectiveness of each intervention was quantified. The main results included effective rate, first urination time, and residual urine volume. CONCLUSION: Objective to provide evidence-based medicine basis for clinicians to choose more effective moxibustion therapy for postoperative urinary retention.

Impacts of aluminum- and iron-based coagulants on municipal sludge anaerobic digestibility, dewaterability, and odor emission.

Although aluminum- and iron-based chemicals have been broadly used as the two most common types of coagulants for wastewater treatment, their impacts on the performance of downstream sludge management can be quite different and have not been well understood. This work reviewed and analyzed their similarities and differences in the context of the anaerobic digestion performance, dewaterability of digested sludge, and odor emission from dewatered biosolids. In short, iron-based coagulants tend to show less negative impact than aluminum-based coagulants. This can be attributed to the reduction of ferric to ferrous ions in the course of anaerobic digestion, which leads to a suite of changes in protein bioavailability, alkalinity and hydrogen sulfide levels, and in turn the sludge dewaterability and odor potential. Whether these observations still hold true in the context of thermally hydrolyzed sludge management remains to be studied. PRACTITIONER POINTS: The impacts of aluminum-/iron-based coagulant addition on municipal sludge anaerobic digestibility, dewaterability, and odor emission are reviewed. Iron-based coagulants show less negative impact on the sludge digestibility than aluminum-based coagulants. Conclusions may aid practitioners in selecting coagulants in practice and better understanding the mechanisms behind the phenomena.

Operational pattern affects nitritation, microbial community and quorum sensing in nitrifying wastewater treatment systems.

Achievement of nitrite accumulation is critical for the application of advanced nitrogen removal processes. Two lab-scale sequencing batch biofilm reactors (SBBRs) and two sequencing batch reactors (SBRs) were operated under intermittent aeration with different feeding patterns. The nitrite accumulation ratio was 56.6% (pulse feeding) and 68.9% (constant feeding) in SBBRs with nitritation. Nitrate accounted for 98% of the effluent nitrogen in SBRs with complete nitrification. The dominant nitrifier was Nitrosomonas in SBBRs and Nitrospira in SBRs. Four types of acyl homoserine lactones (AHLs) were detected. N­[(RS)­3­Hydroxybutyryl]­l­homoserine lactone and N­octanoyl­l­homoserine lactone had a high concentration in the extracellular polymeric substance phase, and had an obvious relationship with nitrite accumulation and ammonia removal. Various microbial communities coexisted in nitrifying systems, with diverse microbial interactions. Microorganisms harboring AHLs-related genes had more interactions with each other, suggesting that nitritation could be regulated by AHLs based quorum sensing.

Effect of anoxic to aerobic duration ratios on nitrogen removal and nitrous oxide emission in the multiple anoxic/aerobic process.

Characteristics of nitrogen removal and nitrous oxide (N2O) emission in the multiple anoxic/aerobic (AO) process were examined in three sequencing batch reactors (SBRs) with different anoxic durations (50 min, SBRH; 40 min, SBRM; 30 min, SBRL) and a fixed aerobic duration of 30 min. The highest total inorganic nitrogen removal percentage of 85.8% was obtained in SBRH, while a minimum N2O emission factor of 1.9% was obtained in SBRL. During nitrification batch experiments, the N2O emission factor and emission rate were both lower in SBRH than SBRL. More N2O production was obtained during denitrification in SBRH when denitrifiers utilized intracellular organic carbon. Nitrite reduction by heterotrophs was the main N2O production pathway during simultaneous nitrification and denitrification in SBRH and SBRL, with the N2O emission factor of 31.3% and 36.3%, respectively. Adequate anoxic duration and lowering aerobic nitrite concentrations could be adopted to mitigate N2O emission in the multiple AO process. The dominant microorganisms at the phylum level in all reactors were Proteobacteria and Bacteroidetes, while the abundance of Nitrospira was the highest in SBRH with relatively lowest dissolved oxygen concentrations.

Autotrophic nitrogen removal in combined nitritation and Anammox systems through intermittent aeration and possible microbial interactions by quorum sensing analysis.

Nitrogen removal and microbial interactions in two combined nitritation and Anammox systems with or without the addition of organics were examined. Two systems were successfully started up by adopting intermittent aeration. Organics addition deteriorated nitrogen removal, and total inorganic nitrogen and ammonium removal percentages decreased by 16.4% and 26.3%, respectively. Organics addition promoted the growth of Chloroflexi and Proteobacteria while suppressed the growth of Candidatus Kuenenia. Organics addition decreased activities of fatty acid biosynthesis and metabolism, amino acid metabolism and biofilm formation, while increased activities of steroid metabolism and glycosaminoglycan biosynthesis. Heterotrophs and Candidatus Kuenenia might interact with other organisms by using diverse quorum sensing systems. Chloroflexi and Proteobacteria interacted with Candidatus Kuenenia in nitrogen metabolism and biofilm formation. Proteobacteria played a key role in building a nitrite loop with Candidatus Kuenenia and nitrifiers. These results clarified microbial interactions in the autotrophic nitrogen removal process and advance its application.

Determination of quorum-sensing signal substances in water and solid phases of activated sludge systems using liquid chromatography-mass spectrometry.

The detection of acyl homoserine lactones (AHLs) in activated sludge is essential for clarifying their function in wastewater treatment processes. An LC-MS/MS method was developed for the detection of AHLs in both the aqueous and solid phases of activated sludge. In addition, the effects of proteases and extracellular polymeric substances (EPS) on the detection of AHLs were evaluated by adding protease inhibitors and extracting EPS, respectively. Recoveries of each AHL were improved by adding 50µL of protease inhibitor, and recoveries were also improved from 0 to 56.9% to 24.2%-105.8% by EPS extraction. Applying the developed method to determine the type and concentration of AHLs showed that C4-HSL, C6-HSL, C8-HSL and 3-oxo-C8-HSL were widely detected in a suspended activated sludge system. The dominant AHL was C8-HSL, with a highest concentration of 304.3ng/L. C4-HSL was mainly distributed in the aqueous phase, whereas C6-HSL, C8-HSL and 3-oxo-C8-HSL were preferentially distributed in the sludge phase.

Potential roles of acyl homoserine lactone based quorum sensing in sequencing batch nitrifying biofilm reactors with or without the addition of organic carbon.

Two lab-scale nitrifying sequencing batch biofilm reactors, with (SBBR_CN) or without the addition of organics (SBBR_N), were operated to investigate potential roles of acyl homoserine lactone (AHL) based quorum sensing. AHLs of N-[(RS)-3-Hydroxybutyryl]-L-homoserine lactone, N-hexanoyl-L-homoserine lactone (C6-HSL) and N-octanoyl-L-homoserine lactone (C8-HSL) were detected in both reactors. C6-HSL and C8-HSL were also detected in batch experiments, especially with stimulated nitrite oxidizing bacteria activities. Quorum sensing affected biofilm formation mainly through the regulation of extracellular protein production. By the metagenomics analysis, many identified genera and species could participate in quorum sensing, quorum quenching and extracellular polymeric substances (EPS) production. A high quorum quenching activity was obtained in SBBR_CN, whereas a high quorum sensing activity in SBBR_N. Nitrosomonas-like ammonia oxidizing bacteria, Nitrospira-like nitrite oxidizing bacteria and Comammox harbored genes for AHL synthesis and EPS production. Possible relationships among AHLs synthesis, biofilm formation and nitrifiers activity were proposed.

Nitrogen removal and nitrous oxide emission from a step-feeding multiple anoxic and aerobic process.

The multiple anoxic and aerobic (AO) process is an advanced biological nitrogen-removal process, and nitrous oxide (N2O) emission might affect its sustainable application. Nitrogen removal and N2O emission in a step-feeding multiple AO sequencing batch reactor (SBRS) was examined, in comparison with a one-feeding sequencing batch reactor (SBRO). Nitrogen removal was enhanced by 12.6% in SBRS compared to the removal percentage of 75.8% in SBRO. Activated sludge in SBRs possessed a higher N2O emission factor during nitrification, denitrification and simultaneous nitrification and denitrification (SND) than in SBRO. A high N2O emission factor was observed during SND in both reactors, with the emission factor of 4.38% in SBRS and 4.66% in SBRO. More N2O emission occurred in the presence of nitrite. Bacteroidetes and Proteobacteria dominated in both SBRS and SBRO. A similar abundance of Thauera, Dechloromonas and Zoogloea possible for denitrification was observed in SBRS and SBRO. Moreover, nosZ from Proteobacteria dominated in both SBRS and SBRO, with dominating genus of Acidovorax, Ralstonia, Thauera and Marinobacter.

[HBx silencing by shRNA down-regulates MMP-2 expression in human hepatocellular carcinoma cell HepG2.2.15].

OBJECTIVE: To observe the effects of HBx shRNA on MMP-2 expression in human hepatocellular carcinoma cell HepG2.2.15. METHODS: HepG2.2.15 cells were transfected with psiHBV/X plasmid using Lipofectamine(TM);2000. The silencing efficacy was evaluated by reverse transcription PCR (RT-PCR) detecting the expression of HBx gene. The proliferation of HepG2.2.15 cells was examined by MTT assay. The expression levels of MMP-2 mRNA and protein were assayed by real-time quantitative PCR (qRT-PCR) and Western blotting, respectively. RESULTS: RT-PCR showed that the efficiency of RNA interference of HBx gene was 53.6%. MTT detection revealed that the cell proliferation was inhibited at 24, 48, and 72 hours after the transfection (0.388±0.087, 0.623±0.016, 0.997±0.036) and the differences had statistically significance as compared with the control group (0.436±0.027, 0.731±0.017, 1.105±0.051) (P<0.05). The qRT-PCR and Western blotting showed that the expression of MMP-2 decreased at both mRNA and protein levels, and the differences had statistically significance as compared with the control group (P<0.05). CONCLUSION: HBx gene RNA interference can inhibit the cell proliferation and down-regulate the expression of MMP-2 in HepG2.2.15 cells.