Impact of influent strengths on nitrous oxide emission and its molecular mechanism in constructed wetlands treating swine wastewater.

Constructed wetlands (CWs) can remove nitrogen (N) through plant assimilation and microbial nitrification and denitrification, while it also releases large greenhouse gas nitrous oxide (N2O) into the atmosphere. However, N2O emissions and the underlying microbial mechanisms of CWs when treating high-strength wastewater have not been systematically surveyed. Here, the effect of three influent strengths on N2O emissions in a pilot-scale CW treatment of swine wastewater was determined and the underlying microbial mechanisms were explored. The results showed that the removal rates of ammonium (NH4+) and total nitrogen (TN) increased significantly with the increasing influent strengths, however, the ratio of N2O emission/TN removal rose by 1.5 times at the same time. Quantitation of microorganisms responsible for N-cycle in the sediment indicated that the abundance of ammonia-oxidizing bacteria (AOB) in high influent strengths (COD, 962.38 ± 3.05 mg/L; NH4+, 317.89 ± 4.24 mg/L) was 51.6-fold compared with that in low influent strengths (COD, 516.94 ± 4.18 mg/L; NH4+, 100.65 ± 2.65), and AOB gradually replaced ammonia-oxidizing archaea (AOA) to dominate ammonia oxidizers. Structural equation models demonstrated that NO2- accumulations promoted the ratio of AOB/AOA, which further led to an increase in the ratio of N2O emission/TN removal. It is worth noting both the N removal rates and N2O emissions increased with the increasing influent strength. To obtain reduced N2O emissions, pretreatment technology for strength reduction should be supplemented before high-strength wastewater enters the CWs. This study may shed new light on the sustainable operation and application of CWs.

Transcriptomic Sequencing and Co-Expression Network Analysis on Key Genes and Pathways Regulating Nitrogen Use Efficiency in Myriophyllum aquaticum.

Massively input and accumulated ammonium is one of the main causes of eutrophication in aquatic ecosystems, which severely deteriorates water quality. Previous studies showed that one of the commonly used macrophytes, Myriophyllum aquaticum, was capable of not only withstanding ammonium of high concentration, but also efficiently assimilating extracellular ammonium to constitutive amino acids and proteins. However, the genetic mechanism regulating such efficient nitrogen metabolism in M. aquaticum is still poorly understood. Therefore, RNA-based analysis was performed in this study to understand the ammonium regulatory mechanism in M. aquaticum in response to various concentrations of ammonium. A total of 7721 genes were differentially expressed, of which those related to nitrogen-transport, assimilation, and remobilization were highly-regulated in response to various concentrations of ammonium. We have also identified transcription factors and protein kinases that were rapidly induced in response to ammonium, which suggests their involvement in ammonium-mediated signalling. Meanwhile, secondary metabolism including phenolics and anthocyanins biosynthesis was also activated in response to various concentrations of ammonium, especially at high ammonium concentrations. These results proposed a complex physiological and genetic regulation network related to nitrogen, carbohydrate, transcription factors, and secondary metabolism for nitrogen use efficiency in M. aquaticum.

Linking N2O Emissions from Biofertilizer-Amended Soil of Tea Plantations to the Abundance and Structure of N2O-Reducing Microbial Communities.

Nitrous oxide (N2O) contributes up to 8% of global greenhouse gas emissions, with approximately 70% from terrestrial sources; over one-third of this terrestrial emission has been linked to increased agricultural fertilizer use. Much of the nitrogen in fertilizers is converted to N2O by microbial processes in soil. However, the potential mechanism of biofertilizers and the role of microbial communities in mitigating soil N2O emissions are not fully understood. Here, we used a greenhouse-based pot experiment with tea plantation soil to investigate the effect of Trichoderma viride biofertilizer on N2O emission. The addition of biofertilizer reduced N2O emissions from fertilized soil by 67.6%. Quantitative PCR (qPCR) analysis of key functional genes involved in N2O generation and reduction ( amoA, nirK, nirS, and nosZ) showed an increased abundance of nirS and nosZ genes linked to the pronounced reduction in N2O emissions. High-throughput sequencing of nosZ showed enhanced relative abundance of nosZ-harboring denitrifiers in the T. viride biofertilizer treatments, thus linking greater N2O reduction capacity to the reduced emissions. Our findings showed that biofertilizers can affect the microbial nitrogen transformation process and reduce N2O emissions from agroecosystems.

Effect of Trichoderma viride biofertilizer on ammonia volatilization from an alkaline soil in Northern China.

Ammonia (NH3) volatilization is one of the primary pathways of nitrogen (N) loss from soils after chemical fertilizer is applied, especially from the alkaline soils in Northern China, which results in lower efficiency for chemical fertilizers. Therefore, we conducted an incubation experiment using an alkaline soil from Tianjin (pH8.37-8.43) to evaluate the suppression effect of Trichoderma viride (T. viride) biofertilizer on NH3 volatilization, and compared the differences in microbial community structure among all samples. The results showed that viable T. viride biofertilizer (T) decreased NH3 volatilization by 42.21% compared with conventional fertilizer ((CK), urea), while nonviable T. viride biofertilizer (TS) decreased NH3 volatilization by 32.42%. NH3 volatilization was significantly higher in CK and sweet potato starch wastewater (SPSW) treatments during the peak period. T. viride biofertilizer also improved the transfer of ammonium from soil to sweet sorghum. Plant dry weights increased 91.23% and 61.08% for T and TS, respectively, compared to CK. Moreover, T. viride biofertilizer enhanced nitrification by increasing the abundance of ammonium-oxidizing archaea (AOA) and ammonium-oxidizing bacteria (AOB). The results of high-throughput sequencing indicated that the microbial community structure and composition were significantly changed by the application of T. viride biofertilizer. This study demonstrated the immense potential of T. viride biofertilizer in reducing NH3 volatilization from alkaline soil and simultaneously improving the utilization of fertilizer N by sweet sorghum.

Isolation of Bdellovibrio bacteriovorus from a tropical wastewater treatment plant and predation of mixed species biofilms assembled by the native community members.

It is reported here that a predatory bacterium belonging to the Genus Bdellovibrio, was isolated from activated sludge at the Ulu Pandan Water Reclamation Plant, Singapore. 16S rDNA gene sequencing analysis revealed that this isolate was 99% identical to 'Bdellovibrio bacteriovorus strain Tiberius' and hence is designated as 'Bdellovibrio bacteriovorus UP'. Using a novel approach based on fluorescence in situ hybridization (FISH), a prey cell density-dependent growth pattern of B. bacteriovorus UP was established. B. bacteriovorus UP preyed upon a broad range of bacterial species (60 species) isolated from the activated sludge. Except for Ochrobactrum anthropi, all Gram-negative species were sensitive to predation by B. bacteriovorus UP irrespective of the mode of growth (planktonic or biofilm). Similarly, the predation-sensitive species were not protected by the predation-resistant species, O. anthropi, as determined in multiple dual-species planktonic and biofilm consortia. Given the broad prey spectrum, B. bacteriovorus UP may impact functional community members, which are largely members of the Proteobacteria. Thus, these results provide an important insight to the role of predatory bacteria in shaping of community structure and function in both natural and engineered ecosystems.

Effects of microbubble pretreatment on physiochemical and microbial properties of excess activated sludge.

Fast increased amount of excess activated sludge (EAS) from wastewater treatment plants has aroused universal concerns on its environmental risks and demands for appropriate treatments, while effective treatment is dependent upon proper pretreatment. In this study, air-supplied microbubbles (air-MBs) with generated size of 25.18 to 28.25 µm were used for EAS pretreatment. Different durations (30, 60, 90, and 120 s) yielded sludge with varied physiochemical conditions, and 60 s decreased sludge oxidation status and significantly increased adenosine triphosphate (ATP) content. Soluble, loosely-bound, and tightly-bound extracellular polymeric substances (SEPS, LB-EPS, and TB-EPS) were extracted from the sludge through a stepwise approach and examined through three-dimensional excitation-emission matrix (3D-EEM) and quantitative analysis. The results showed that 60- and 120-s treatments generated stronger fluorescence intensities on dissolved organic matters (DOMs) of protein-like and fulvic acid in LB-EPS and TB-EPS, which indicated the decrease of counterparts in EAS, and therefore facilitated sludge dewaterability and reduction. The dominant microbial communities in EAS, including Proteobacteria, Bacteroidota, Chloroflexi, and Actinobacteriota, were not significantly affected by MB pretreatment. The results collectively revealed the effects of MB pretreatment on EAS and indicated that MBs could be an effective pretreatment technique for EAS treatment process.

Complex regulatory network allows Myriophyllum aquaticum to thrive under high-concentration ammonia toxicity.

Plants easily experience ammonia (NH4+) toxicity, especially aquatic plants. However, a unique wetland plant species, Myriophyllum aquaticum, can survive in livestock wastewater with more than 26 mM NH4+. In this study, the mechanisms of the M. aquaticum response to NH4+ toxicity were analysed with RNA-seq. Preliminary analysis of enzyme activities indicated that key enzymes involved in nitrogen metabolism were activated to assimilate toxic NH4+ into amino acids and proteins. In response to photosystem damage, M. aquaticum seemed to remobilize starch and cellulose for greater carbon and energy supplies to resist NH4+ toxicity. Antioxidative enzyme activity and the secondary metabolite content were significantly elevated for reactive oxygen species removal. Transcriptomic analyses also revealed that genes involved in diverse functions (e.g., nitrogen, carbon and secondary metabolisms) were highly responsive to NH4+ stress. These results suggested that a complex physiological and genetic regulatory network in M. aquaticum contributes to its NH4+ tolerance.

Achieving efficient nitrogen removal from real sewage via nitrite pathway in a continuous nitrogen removal process by combining free nitrous acid sludge treatment and DO control.

The incomplete denitrification due to insufficient carbon resource in the wastewater treatment plants (WWTPs) resulted in low nitrogen removal efficiency, which has become a widespread problem in China and all around the world. Reducing the requirement of carbon source by manipulating the nitrogen removal pathway from conventional nitrification-denitrification to partial nitrification-denitrification is considered as an efficient solution. In this article, the feasibility of combining free nitrous acid (FNA) sludge treatment and DO control to achieve partial nitrification-denitrification in a continuous flow system (aerobic-anoxic-oxic process) using real sewage was assessed. The nitrite pathway was rapidly established in the experimental reactor within 23 days by simultaneously lowering DO concentration in aerobic zone to 0.5 mg/L and treating 30% of the activated sludge per day from the reactor in the FNA sludge treatment unit with FNA concentration of 1.2 mg N/L and exposure time of 18 h. The nitrite oxidizing bacteria (NOB) were efficiently washed out and the partial nitrification process could maintain stable in the experimental reactor even after cease of FNA treatment and increase of DO concentrations in the main stream to 1.5 mg/L, with an average nitrite accumulation rate of above 78%. In contrast, the nitrite accumulation rate was just around 58% during low DO concentrations phase and declined quickly to below 1% after the DO concentrations were increased to 1.5 mg/L in the control reactor which only utilized single strategy of DO control to achieve nitrite pathway. Moreover, a better sludge settleability and nitrogen removal performance could also be realized in the experimental reactor. The results of nitrifying bacteria activities and quantities detection demonstrated that although NOB activities in both reactors were effectively inhibited, a certain amount of NOB (6.26 × 106 copies/g MLSS) were remained in the control reactor and multiplied rapidly as the DO concentration increased, which might break down the partial nitrification. Furthermore, the quantity results of nitrogen cycling related functional genes showed that the increment of the ratio of nitrate reduced bacteria to total bacteria was 0.35% larger than that of nitric oxide bacteria in the control reactor, while those two ratios increased similarly by 1.11% and 1.12% in the experimental reactor, respectively, which might be one potential cause of reduction in N2O emission of nitrite pathway achieved by FNA-based technologies.

Predation by Bdellovibrio bacteriovorus significantly reduces viability and alters the microbial community composition of activated sludge flocs and granules.

We recently isolated and characterised a predatory Bdellovibrio bacteriovorus strain from activated sludge (Ulu Pandan Water Reclamation Plant, Singapore), and this strain, B. bacteriovorus UP, was able to prey upon a broad spectrum of bacterial isolates from the activated sludge when grown as planktonic cells or as biofilms. Here, we have tested the effect of Bdellovibrio predation on floccular and granular sludge to determine if the spatial organisation, loosely or tightly aggregated communities, was protective from predation. The effect of predation was assessed using a combination of biomass quantification, cellular activity measurement and microscopic image analysis to determine community viability. Additionally, changes in the microbial communities due to predation by B. bacteriovorus UP were analysed through total RNA sequencing. Predation led to a significant reduction in microbial activity and total biomass for both floccular and granular sludge communities. Predation was also associated with significant changes in the microbial community composition in both communities, with >90% of the community members reduced in relative abundance after 24 h. Of those community members, the dominant organisms, such as Proteobacteria and Bacteroidetes, were the most affected phylotypes. This suggests that predatory bacteria, which display indiscriminant feeding, could significantly shift the species composition and thus, may disturb the operational performance of wastewater treatment systems.