Carbon flow, energy metabolic intensity and metagenomic characteristics of a Fe (III)-enhanced anerobic digestion system during treating swine wastewater.

Deep treatment and bioenergy recovery of swine wastewater (SW) are beneficial for constructing a low-carbon footprint and resource-recycling society. In this study, Fe (III) addition from 0 to 600 mg/L significantly increased the methane (CH4) content of the recovered biogas from 61.4 ± 2.0 to 89.3 ± 2.0 % during SW treatment in an anaerobic membrane digestion system. The specific methane yields (SMY) also increased significantly from 0.20 ± 0.05 to 0.29 ± 0.02 L/g COD. Fe (III) and its bio-transformed products which participated in establishing direct interspecific electron transfer (DIET), upregulated the abundance of e-pili and Nicotinamide adenine dinucleotide (NADH), enriched electroactive bacteria. The increase in cellular adenosine triphosphate (cATP) from 6583 to 14,518 ng/gVSS and electron transport system (ETS) from 1468 to 1968 mg/(g·h) promoted the intensity of energy flow and electron flow during anaerobic digestion of SW. Moreover, Fe (III) promoted the hydrolysis and acidification of organic matters, and strengthened the acetoacetic methanogenesis pathway. This study established an approach for harvesting high quality bioenergy from SW and revealed the effects and mechanisms from the view of carbon flow, energy metabolic intensity and metagenomics.

Potential electron acceptors for ammonium oxidation in wastewater treatment system under anoxic condition: A review.

Anaerobic ammonium oxidation has been considered as an environmental-friendly and energy-efficient biological nitrogen removal (BNR) technology. Recently, new reaction pathway for ammonium oxidation under anaerobic condition had been discovered. In addition to nitrite, iron trivalent, sulfate, manganese and electrons from electrode might be potential electron acceptors for ammonium oxidation, which can be coupled to traditional BNR process for wastewater treatment. In this paper, the pathway and mechanism for ammonium oxidation with various electron acceptors under anaerobic condition is studied comprehensively, and the research progress of potentially functional microbes is summarized. The potential application of various electron acceptors for ammonium oxidation in wastewater is addressed, and the N2O emission during nitrogen removal is also discussed, which was important greenhouse gas for global climate change. The problems remained unclear for ammonium oxidation by multi-electron acceptors and potential interactions are also discussed in this review.

Respiration and growth of Paracoccus denitrificans R-1 with nitrous oxide as an electron acceptor.

In the nitrogen biogeochemical cycle, the reduction of nitrous oxide (N2O) to N2 by N2O reductase, which is encoded by nosZ gene, is the only biological pathway for N2O consumption. In this study, we successfully isolated a strain of denitrifying Paracoccus denitrificans R-1 from sewage treatment plant sludge. This strain has strong N2O reduction capability, and the average N2O reduction rate was 5.10 ± 0.11 × 10-9 µmol·h-1·cell-1 under anaerobic condition in a defined medium. This reduction was accompanied by the stoichiometric consumption of acetate over time when N2O served as the sole electron acceptor and the reduction can yield energy to support microbial growth, suggesting that microbial N2O reduction is related to the energy generation process. Genomic analysis showed that the gene cluster encoding N2O reductase of P. denitrificans R-1 was composed of nosR, nosZ, nosD, nosF, nosY, nosL, and nosZ, which was identified as that in other strains in clade I. Respiratory inhibitors test indicated that the pathway of electron transport for N2O reduction was different from that of the traditional electron transport chain for aerobic respiration. Cu2+, silver nanoparticles, O2, and acidic conditions can strongly inhibit the reduction, whereas NO3- or NH4+ can promote it. These findings suggest that modular N2O reduction of P. denitrificans R-1 is linked to the electron transport and energy conservation, and dissimilatory N2O reduction is a form of microbial anaerobic respiration. IMPORTANCE: Nitrous oxide (N2O) is a potent greenhouse gas and contributor to ozone layer destruction, and atmospheric N2O has increased steadily over the past century due to human activities. The release of N2O from fixed N is almost entirely controlled by microbial N2O reductase activities. Here, we investigated the ability to obtain energy for the growth of Paracoccus denitrificans R-1 by coupling the oxidation of various electron donors to N2O reduction. The modular N2O reduction process of denitrifying microorganism not only can consume N2O produced by itself but also can consume the external N2O generated from biological or abiotic pathways under suitable condition, which should be critical for controlling the release of N2O from ecosystems into the atmosphere.

Ferric and sulfate coupled ammonium oxidation enhanced nitrogen removal in two-stage partial nitrification - Anammox/denitrification process for food waste liquid digestate treatment.

Liquid digestate of food waste is an ammonium-, ferric- and sulfate-laden leachate produced during digestate dewatering, where the carbon source is insufficient for nitrogen removal. A two-stage partial nitrification-anammox/denitrification process was established for nitrogen removal of liquid digestate without pre-treatment (>300 d), through which nitrogen (95 %), biodegradable organics (100 %), sulfate (78 %) and iron (100 %) were efficiently removed. Additional ammonium conversion (20 %N) might be coupled with ferric and sulfate reduction, while produced nitrite could be further converted to di-nitrogen gas through anammox (75 %) and denitrification (25 %). Notably, since increasingly contribution of hydroxylamine producing nitrous oxide, and up-regulated expression of electron transfer and cytochrome c protein, the enhanced ammonium oxidation was probably conducted through extracellular polymeric substances-mediated electron transfer between sulfate/ferric-reducers and aerobic ammonium oxidizers. Thus, the established partial nitrification-anammox/denitrification process might be a cost-efficient nitrogen removal technology for liquid digestate, benefitting to domestic waste recycling and carbon neutralization.

Micro-aeration and low influent C/N are key environmental factors for achieving ANAMMOX in livestock farming wastewater treatment plants.

Anaerobic ammonium oxidation (ANAMMOX)-mediated system is a cost-effective green nitrogen removal process. However, there are few examples of successful application of this advanced wastewater denitrification process in wastewater treatment plants, and the understanding of how to implement anaerobic ammonia oxidation process in full-scale is still limited. In this study, it was found that the abundance of anaerobic ammonia-oxidizing bacteria (AnAOB) in the two livestock wastewater plants named J1 and J2, respectively, showed diametrically opposed trends of waxing and waning with time. The microbial communities of the activated sludge in the two plants at different time were sampled and analyzed by high-throughput sequencing of 16S rRNA genes. Structural equation models (SEMs) were used to reveal the key factors affecting the realization of the ANAMMOX. Changes in the concentration of dissolved oxygen and C/N had a significant effect on the relative abundance of anaerobic ammonia oxidation bacteria (AnAOB). The low concentration of DO (0.2∼0.5 mg/L) could inhibit the activity of nitrifying bacteria (NOB) to achieve partial oxidation of ammonia nitrogen and provide sufficient substrate for the growth of AnAOB, similar to the CANON (Completely Autotrophic Nitrogen removal Over Nitrite). Unlike CANON, heterotrophic denitrification is also a particularly critical part of the livestock wastewater treatment, and a suitable C/N of about 0.6 could reduce the competition risk of heterotrophic microorganisms to AnAOB and ensure a good ecological niche for AnAOB. Based on the results of 16S rRNA and microbial co-occurrence networks, it was discovered that microorganisms in the sludge not only had a richer network interaction, but also achieved a mutually beneficial symbiotic interaction network among denitrifying bacteria (Pseudomonas sp., Terrimonas sp., Dokdonella sp.), AnAOB (Candidatus Brocadia sp.) at DO of 0.2∼0.5 mg/L and C/N of 0.6. Among the top 20 in abundance of genus level, AnAOB had a high relative abundance of 27.66%, followed by denitrifying bacteria of 3.67%, AOB of 0.64% and NOB of 0.26%, which is an essential indicator for the emergence of an AnAOB-dominated nitrogen removal cycle. In conclusion, this study highlights the importance of dissolved oxygen and C/N regulation by analyzing the mechanism of ANAMMOX sludge extinction and growth in two plants under anthropogenic regulation of AnAOB in full-scale wastewater treatment systems.

A critical review of exogenous additives for improving the anammox process.

Anammox achieves chemoautotrophic nitrogen removal under anaerobic and anoxic conditions and is a low-carbon wastewater biological nitrogen removal process with broad application potential. However, the physiological limitations of AnAOB often cause problems in engineering applications, such as a long start-up time, unstable operation, easily inhibited reactions, and difficulty in long-term strain preservation. Exogenous additives have been considered an alternative strategy to address these issues by retaining microbes, shortening the doubling time of AnAOB and improving functional enzyme activity. This paper reviews the role of carriers, biochar, intermediates, metal ions, reaction substrates, redox buffers, cryoprotectants and organics in optimizing anammox. The pathways and mechanisms of exogenous additives, which are explored to solve problems, are systematically summarized and analyzed in this article according to operational performance, functional enzyme activity, and microbial abundance to provide helpful information for the engineering application of anammox.

A mixed blessing of viruses in wastewater treatment plants.

Activated sludge of wastewater treatment plants harbors a very high diversity of both microorganisms and viruses, wherein the latter control microbial dynamics and metabolisms by infection and lysis of cells. However, it remains poorly understood how viruses impact the biochemical processes of activated sludge, for example in terms of treatment efficiency and pollutant removal. Using metagenomic and metatranscriptomic deep sequencing, the present study recovered thousands of viral sequences from activated sludge samples of three conventional wastewater treatment plants. Gene-sharing network indicated that most of viruses could not be assigned to known viral genera, implying activated sludge as an underexplored reservoir for new viruses and viral diversity. In silico predictions of virus-host linkages demonstrated that infected microbial hosts, mostly belonging to bacteria, were transcriptionally active and able to hydrolyze polymers including starches, celluloses, and proteins. Some viruses encode auxiliary metabolic genes (AMGs) involved in carbon, nitrogen, and sulfur cycling, and antibiotic resistance genes (ARGs) for resistance to multiple drugs. The virus-encoded AMGs may enhance the biodegradation of contaminants like starches and celluloses, suggesting a positive role for viruses in strengthening the performance of activated sludge. However, ARGs would be disseminated to different microorganisms using viruses as gene shuttles, demonstrating the possibility for viruses to facilitate the spread of antibiotic resistance in the environment. Collectively, this study highlights the mixed blessing of viruses in wastewater treatment plants, and deciphers how they manipulate the biochemical processes in the activated sludge, with implications for both environmental protection and ecosystem security.

Nitrification mainly driven by ammonia-oxidizing bacteria and nitrite-oxidizing bacteria in an anammox-inoculated wastewater treatment system.

Anaerobic ammonium oxidation (anammox) process has been acknowledged as an environmentally friendly and time-saving technique capable of achieving efficient nitrogen removal. However, the community of nitrification process in anammox-inoculated wastewater treatment plants (WWTPs) has not been elucidated. In this study, ammonia oxidation (AO) and nitrite oxidation (NO) rates were analyzed with the incubation of activated sludge from Xinfeng WWTPs (Taiwan, China), and the community composition of nitrification communities were investigated by high-throughput sequencing. Results showed that both AO and NO had strong activity in the activated sludge. The average rates of AO and NO in sample A were 6.51 µmol L-1 h-1 and 6.52 µmol L-1 h-1, respectively, while the rates in sample B were 14.48 µmol L-1 h-1 and 14.59 µmol L-1 h-1, respectively. The abundance of the nitrite-oxidizing bacteria (NOB) Nitrospira was 0.89-4.95 × 1011 copies/g in both samples A and B, the abundance of ammonia-oxidizing bacteria (AOB) was 1.01-9.74 × 109 copies/g. In contrast, the abundance of ammonia-oxidizing archaea (AOA) was much lower than AOB, only with 1.28-1.53 × 105 copies/g in samples A and B. The AOA community was dominated by Nitrosotenuis, Nitrosocosmicus, and Nitrososphaera, while the AOB community mainly consisted of Nitrosomonas and Nitrosococcus. The dominant species of Nitrospira were Candidatus Nitrospira defluvii, Candidatus Nitrospira Ecomare2 and Nitrospira inopinata. In summary, the strong nitrification activity was mainly catalyzed by AOB and Nitrospira, maintaining high efficiency in nitrogen removal in the anammox-inoculated WWTPs by providing the substrates required for denitrification and anammox processes.

Biogas energy generated from livestock manure in China: Current situation and future trends.

This article investigates the current status of the livestock industry (cattle, pigs, sheep, and poultry) in China and assesses the potential for biogas production from anaerobically digested livestock manure. According to calculation results based on the latest data of livestock released by the National Bureau of Statistics of China in 2018, China produced 2 × 1012 kg of manure pollution in 2017, with pig waste representing the largest single manure source. Biogas that can be converted from high organic containing manure is a kind of clean bioenergy with low carbon footprint. In 2017, the energy potential from manure-produced biogas was about 5.74 × 1012-6.73 × 1012 MJ, which corresponds to 4-5% of China's total energy demand. Correlation analysis between biogas production and the livestock industry showed that crop production had significant effects on manure-generated biogas production. However, it is necessary to address the challenges when applying AD technology. Bioenergy potential from manure will be lost during material collection and transportation. Although large-scale livestock farming remains controversial, this type of farming can improve the energy recovery rate of livestock manure. How to gain benefits and maintain sustainable development is also a bottleneck for AD promotion. Reducing energy input in AD projects as well as enhancing the efficiency of methanogenesis of livestock manure are key factors for achieving a high net output of biogas projects. More inclusive strategies and a broader vision should be adopted to allow stakeholders to benefit from manure-generated biogas projects.

Survival strategies of ammonia-oxidizing archaea (AOA) in a full-scale WWTP treating mixed landfill leachate containing copper ions and operating at low-intensity of aeration.

Recent studies indicate that ammonia-oxidizing archaea (AOA) may play an important role in nitrogen removal by wastewater treatment plants (WWTPs). However, our knowledge of the mechanisms employed by AOA for growth and survival in full-scale WWTPs is still limited. Here, metagenomic and metatranscriptomic analyses combined with a laboratory cultivation experiment revealed that three active AOAs (WS9, WS192, and WS208) belonging to family Nitrososphaeraceae were active in the deep oxidation ditch (DOD) of a full-scale WWTP treating landfill leachate, which is configured with three continuous aerobic-anoxic (OA) modules with low-intensity aeration (≤ 1.5 mg/L). AOA coexisted with AOB and complete ammonia oxidizers (Comammox), while the ammonia-oxidizing microbial (AOM) community was unexpectedly dominated by the novel AOA strain WS9. The low aeration, long retention time, and relatively high inputs of ammonium and copper might be responsible for the survival of AOA over AOB and Comammox, while the dominance of WS9, specifically may be enhanced by substrate preference and uniquely encoded retention strategies. The urease-negative WS9 is specifically adapted for ammonia acquisition as evidenced by the high expression of an ammonium transporter, whereas two metabolically versatile urease-positive AOA strains (WS192 and WS208) can likely supplement ammonia needs with urea. This study provides important information for the survival and application of the eutrophic Nitrososphaeraceae AOA and advances our understanding of archaea-dominated ammonia oxidation in a full-scale wastewater treatment system.

Nanoarchitectured structure and population dynamics of anaerobic ammonium oxidizing (anammox) bacteria in a wastewater treatment plant.

Studies on microbial community and population dynamics are essential for the successful development, monitoring and operation of biological wastewater treatment systems. Especially for novel or sustainable systems such as the anaerobic ammonium oxidizing (anammox) process that are not yet well explored. Here we collected granular microbial sludge samples and investigated a community of anammox bacteria over a period of four years, divided into eight stages in a full scale simultaneous partial nitrification, anammox and denitrification (SNAD) process for treating landfill leachate. Specific qPCR primers were designed to target and quantify the two most abundant anammox species, Candidatus Kuenenia stuttgartiensis (KS) and Candidatus Brocadia anammoxidans (BA). The two species were monitored and could explain the dynamic shift of the anammox community corresponding to the operating conditions. Using the newly designed KS-specific primer (KSqF3/KSqR3) and BA-specific primer (BAqF/BAqR), we estimated the amounts of KS and BA to be in the range of 6.2 × 106 to 5.9 × 108 and 1.1 × 105 to 4.1 × 107 copies µg-1 DNA, respectively. KS was found to be the dominant species in all anammox granules studied and played an important role in the formation of granules. The KS/BA ratio was positively correlated to the size of granules in the reactor and ammonia nitrogen removal efficiency of the treatment plant.

Specific and effective detection of anammox bacteria using PCR primers targeting the 16S rRNA gene and functional genes.

Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the nitrogen cycle by coupling ammonium and nitrite to produce dinitrogen gas (N2). Polymerase chain reaction (PCR) is a fast, simple, and sensitive method that is widely used to assess the diversity, abundance, and activity of the slow-growing bacteria. In this review, we summarize and evaluate the wide variety of PCR primers targeting the 16S rRNA gene and functional genes (hzo, nir, and hzs) of anammox bacteria for their effectiveness and efficiencies in detecting this group of bacteria in different sample types. Furthermore, the efficiencies of different universal high-throughput sequencing 16S rRNA gene primers in anammox bacteria investigations were also evaluated to provide a reference for primer selection. Based on our in silico evaluation results, none of the 16S rRNA gene primers could recover all of the known anammox bacteria, but multiple hzo and hzs gene primers could accomplish this task. However, uncertain copies (1-3 copies) of hzo genes were identified in the genomes, and the hydrazine oxidation reaction catalyzed by hydrazine oxidoreductases (HZOs) can also be catalyzed by other hydroxylamine oxidoreductases (HAOs) in anammox bacteria, which can potentially result in large deviations in hzo-based qPCR and RT-qPCR analyses and results. Therefore, the use of optimal primers targeting unique hzs genes are recommended, although the efficiencies of these newly designed primers need further verification in practical applications. This article provides comprehensive information for the effective and specific detection of anammox bacteria using specific primers targeting the 16S rRNA gene and functional genes and serves as a basis for future high-quality primer design.

Activity and Metabolic Versatility of Complete Ammonia Oxidizers in Full-Scale Wastewater Treatment Systems.

The recent discovery of complete ammonia oxidizers (comammox) contradicts the paradigm that chemolithoautotrophic nitrification is always catalyzed by two different microorganisms. However, our knowledge of the survival strategies of comammox in complex ecosystems, such as full-scale wastewater treatment plants (WWTPs), remains limited. Analyses of genomes and in situ transcriptomes of four comammox organisms from two full-scale WWTPs revealed that comammox were active and showed a surprisingly high metabolic versatility. A gene cluster for the utilization of urea and a gene encoding cyanase suggest that comammox may use diverse organic nitrogen compounds in addition to free ammonia as the substrates. The comammox organisms also encoded the genomic potential for multiple alternative energy metabolisms, including respiration with hydrogen, formate, and sulfite as electron donors. Pathways for the biosynthesis and degradation of polyphosphate, glycogen, and polyhydroxyalkanoates as intracellular storage compounds likely help comammox survive unfavorable conditions and facilitate switches between lifestyles in fluctuating environments. One of the comammox strains acquired from the anaerobic tank encoded and transcribed genes involved in homoacetate fermentation or in the utilization of exogenous acetate, both pathways being unexpected in a nitrifying bacterium. Surprisingly, this strain also encoded a respiratory nitrate reductase which has not yet been found in any other Nitrospira genome and might confer a selective advantage to this strain over other Nitrospira strains in anoxic conditions.IMPORTANCE The discovery of comammox in the genus Nitrospira changes our perception of nitrification. However, genomes of comammox organisms have not been acquired from full-scale WWTPs, and very little is known about their survival strategies and potential metabolisms in complex wastewater treatment systems. Here, four comammox metagenome-assembled genomes and metatranscriptomic data sets were retrieved from two full-scale WWTPs. Their impressive and-among nitrifiers-unsurpassed ecophysiological versatility could make comammox Nitrospira an interesting target for optimizing nitrification in current and future bioreactor configurations.

Complex microbial nitrogen-cycling networks in three distinct anammox-inoculated wastewater treatment systems.

Microbial nitrogen removal mediated by anaerobic ammonium oxidation (anammox) are cost-effective, yet it is time-consuming to accumulate the slow-growing anammox bacteria in conventional wastewater treatment plants (WWTPs). Inoculation of anammox enriched pellets is an effective way to establish anammox and achieve shortcut nitrogen removal in full-scale WWTPs. However, little is known about the complex microbial nitrogen-cycling networks in these anammox-inoculated WWTPs. Here, we applied metagenomic and metatranscriptomic tools to study the microbial nitrogen removal in three conventional WWTPs, which have been inoculated exogenous anammox pellets, representing partial-nitrification anammox (PNA) and nitrification-denitrification nitrogen removal processes. In the PNA system of Bali (BL), ammonia was partially oxidized by ammonia-oxidizing bacteria (AOB) Nitrosomonas and the oxidized nitrite and the remaining ammonium were directly converted to N2 by anammox bacteria Ca. Brocadia and Ca. Kuenenia. In the nitrification-denitrification system of Wenshan (WS), ammonia-oxidizing archaea (AOA) Thaumarchaeota unexpectedly dominated the nitrifying community in the presence of AOB Nitrosomonas. Meanwhile, the biomass yield of Ca. Brocadia was likely inhibited by the high biodegradable organic compound input and limited by substrate competitions from AOA, AOB, complete ammonia oxidizers (comammox) Nitrospira, nitrite-oxidizing bacteria (NOB) Nitrospira, and heterotrophic denitrifiers. Unexpectedly, comammox Nitrospira was the predominant nitrifier in the presence of AOB Nitrosomonas in the organic carbon-rich nitrification-denitrification system of Linkou (LK). These results clearly showed that distinct active groups were working in concert for an effective nitrogen removal in different WWTPs. This study confirmed the feasibility of anammox application in ammonium-rich systems by direct inoculation of the exogenous anammox pellets and improved our understanding of microbial nitrogen cycling in anammox-driven conventional WWTPs from both physiochemical and omics perspectives.

A xylan-degrading thermophilic and obligate anaerobe Xylanivirga thermophila gen. nov., sp. nov., isolated from an anammox dominant wastewater treatment plant, and proposal of Xylanivirgaceae fam. nov.

In our search for novel anaerobes with potential carbohydrate polymers degrading activity, we have isolated a xylan-degrading bacterial strain SYSU GA17129T from an anammox bacteria dominant wastewater treatment plant. Phylogenetic analysis of the 16S rRNA gene sequence indicated the strain SYSU GA17129T belong to the order Clostridiales and shared highest sequence identity with Caldicoprobacter faecalis DSM 20678T (89.9%). The strain was thermophilic, obligately anaerobic, non-motile and rod shaped. Optimum growth of the strain was observed at 45 °C, pH 8.0 and in the presence of 0.5% NaCl (w/v). The chemotaxonomic features of the strain SYSU GA17129T comprised of C14:0 FAME, iso-C15:0 FAME and C16:0 FAME as the major fatty acids (>10%), diphosphatidylglycerol, phosphatidylinositol mannoside, an unidentified phospholipid, three unidentified polar lipids and two unidentified glycolipids as its polar lipids, and meso-diaminopimelic acid (meso-DAP) as the diamino acid in peptidoglycan. The G + C content of the genomic DNA was 35.9%. The strain could be distinguished from other defined families within the order Clostridiales by the differences in phenotypic and physiological characteristics, distinct phylogenetic lineage in 16S rRNA gene- and genome-based phylogenies and low genomic relatedness index. Based on these distinguishing properties, strain SYSU GA17129T is proposed to represent a new species of a new genus Xylanivirga thermophila gen. nov., sp. nov., within a new family Xylanivirgaceae fam. nov. The type species of the new taxon is SYSU GA17129T (=KCTC 15754T = CGMCC 1.5282T). This strain is characterized within the order Clostridiales, class Clostridia of the phylum Firmicutes.

The short- and long-term inhibitory effects of Fe (II) on anaerobic ammonium oxidizing (anammox) process.

The application of the anammox process has great potential in treating nitrogen-rich wastewater. The presence of Fe (II) is expected to affect the growth and activity of anammox bacteria. Short-term (acute) and long-term effects (chronic) of Fe (II) on anammox activity were investigated. In the short-term study, results demonstrated that the optimum concentration of Fe (II) that could be added to anammox is 0.08 mM, at which specific anammox activity (SAA) improved by 60% compared to the control assay, 0.00 mM. The inhibition concentration, IC50, of Fe (II) was found to be 0.192 mM. Kinetics of anammox specific growth rate were estimated based on results of the batch test and evaluated with Han-Levenspiel's substrate inhibition kinetics model. The optimum concentration and IC50 of Fe (II) predicted by the Han-Levenspiel model was similar to the batch test, with values of 0.07 mM and 0.20 mM, respectively. The long-term effect of Fe (II) on the performance of a sequencing batch reactor (SBR) was evaluated. Results showed that an appropriate Fe (II) addition enhanced anammox activity, achieving 85% NH4 +-N and 96% NO2 --N removal efficiency when 0.08 mM of Fe (II) was added. Quantitative polymerase chain reaction (qPCR) was adopted to detect and identify the anammox bacteria.

Metagenomic and metatranscriptomic analyses reveal activity and hosts of antibiotic resistance genes in activated sludge.

Wastewater treatment plants (WWTPs) are a source and reservoir for subsequent spread of various antibiotic resistance genes (ARGs). However, little is known about the activity and hosts of ARGs in WWTPs. Here, we utilized both metagenomic and metatranscriptomic approaches to comprehensively reveal the diversity, abundance, expression and hosts of ARGs in activated sludge (AS) from three conventional WWTPs in Taiwan. Based on deep sequencing data and a custom-made ARG database, a total of 360 ARGs associated with 24 classes of antibiotics were identified from the three AS metagenomes, with an abundance range of 7.06 × 10-1-1.20 × 10-4 copies of ARG/copy of 16S rRNA gene. Differential coverage binning analysis revealed that >22 bacterial phyla were the putative hosts of the identified ARGs. Surprisingly, genus Mycobacterium and family Burkholderiaceae were observed as multi-drug resistant harboring 14 and 50 ARGs. Metatranscriptome analysis showed 65.8% of the identified ARGs were being expressed, highlighting that ARGs were not only present, but also transcriptionally active in AS. Remarkably, 110 identified ARGs were annotated as plasmid-associated and displayed a close to two-fold increased likelihood of being transcriptionally expressed compared to those ARGs found exclusively within bacterial chromosomes. Further analysis showed the transcript abundance of aminoglycoside, sulfonamide, and tetracycline resistance genes was mainly contributed by plasmid-borne ARGs. Our approach allowed us to specifically link ARGs to their transcripts and genetic context, providing a comprehensive insight into the prevalence, expression and hosts of ARGs in AS. Overall, results of this study enhance our understanding of the distribution and dissemination of ARGs in WWTPs, which benefits environmental risk assessment and management of ARB and ARGs.

Assessment of molecular detection of anaerobic ammonium-oxidizing (anammox) bacteria in different environmental samples using PCR primers based on 16S rRNA and functional genes.

Eleven published PCR primer sets for detecting genes encoding 16S ribosomal RNA (rRNA), hydrazine oxidoreductase (HZO), cytochrome cd 1-containing nitrite reductase (NirS), and hydrazine synthase subunit A (HzsA) of anaerobic ammonium-oxidizing (anammox) bacteria were assessed for the diversity and abundance of anammox bacteria in samples of three environments: wastewater treatment plant (WWTP), wetland of Mai Po Nature Reserve (MP), and the South China Sea (SCS). Consistent phylogenetic results of three biomarkers (16S rRNA, hzo, and hzsA) of anammox bacteria were obtained from all samples. WWTP had the lowest diversity with Candidatus Kuenenia dominating while the SCS was dominated by Candidatus Scalindua. MP showed the highest diversity of anammox bacteria including C. Scalindua, C. Kuenenia, and Candidatus Brocadia. Comparing different primer sets, no significant differences in specificity for 16S rRNA gene could be distinguished. Primer set CL1 showed relatively high efficiency in detecting the anammox bacterium hzo gene from all samples, while CL2 showed greater selectivity for WWTP samples. The recently reported primer sets of the hzsA gene resulted in high efficiencies in detecting anammox bacteria while nirS primer sets were more selective for specific samples. Results collectively indicate that the distribution of anammox bacteria is niche-specific within different ecosystems and primer specificity may cause biases on the diversity detected.

Current advances in molecular methods for detection of nitrite-dependent anaerobic methane oxidizing bacteria in natural environments.

Nitrite-dependent anaerobic methane oxidation (n-damo) process uniquely links microbial nitrogen and carbon cycles. Research on n-damo bacteria progresses quickly with experimental evidences through enrichment cultures. Polymerase chain reaction (PCR)-based methods for detecting them in various natural ecosystems and engineered systems play a very important role in the discovery of their distribution, abundance, and biodiversity in the ecosystems. Important characteristics of n-damo enrichments were obtained and their key significance in microbial nitrogen and carbon cycles was investigated. The molecular methods currently used in detecting n-damo bacteria were comprehensively reviewed and discussed for their strengths and limitations in applications with a wide range of samples. The pmoA gene-based PCR primers for n-damo bacterial detection were evaluated and, in particular, several incorrectly stated PCR primer nucleotide sequences in the published papers were also pointed out to allow correct applications of the PCR primers in current and future investigations. Furthermore, this review also offers the future perspectives of n-damo bacteria based on current information and methods available for a better acquisition of new knowledge about this group of bacteria.

Biodegradation of tetramethylammonium hydroxide (TMAH) in completely autotrophic nitrogen removal over nitrite (CANON) process.

This study conducted a completely autotrophic nitrogen removal over nitrite (CANON) process in a continuous anoxic upflow bioreactor to treat synthetic wastewater with TMAH (tetramethylammonium hydroxide) ranging from 200 to 1000mg/L. The intermediates were analyzed for understanding the metabolic pathway of TMAH biodegradation in CANON process. In addition, (15)N-labeled TMAH was used as the substrate in a batch anoxic bioreactor to confirm that TMAH was converted to nitrogen gas in CANON process. The results indicated that TMAH was almost completely biodegraded in CANON system at different influent TMAH concentrations of 200, 500, and 1000mg/L. The average removal efficiencies of total nitrogen were higher than 90% during the experiments. Trimethylamine (TMA) and methylamine (MA) were found to be the main biodegradation intermediates of TMAH in CANON process. The production of nitrogen gas with (15)N-labeled during the batch anaerobic bioreactor indicated that CANON process successfully converted TMAH into nitrogen gas.