Reactivated biofilm coupling n-DAMO with anammox achieved high-rate nitrogen removal in membrane aerated moving bed biofilm reactor.

As a promising technology, the combination of nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) with Anammox offers a solution to achieve effective and sustainable wastewater treatment. However, this sustainable process faces challenges to accumulate sufficient biomass for reaching practical nitrogen removal performance. This study developed an innovative membrane aerated moving bed biofilm reactor (MAMBBR), which supported sufficient methane supply and excellent biofilm attachment, for cultivating biofilms coupling n-DAMO with Anammox. Biofilms were developed rapidly on the polyurethane foam with the supply of ammonium and nitrate, achieving the bioreactor performance of 275 g N m-3 d-1 within 102 days. After the preservation at -20 °C for 8 months, the biofilm was successfully reactivated and achieved 315 g N m-3 d-1 after 188 days. After reactivation, MAMBBR was applied to treat synthetic sidestream wastewater. Up to 99.9% of total nitrogen was removed with the bioreactor performance of 4.0 kg N m-3 d-1. Microbial community analysis and mass balance calculation demonstrated that n-DAMO microorganisms and Anammox bacteria collectively contributed to nitrogen removal in MAMBBR. The MAMBBR developed in this study provides an ideal system of integrating n-DAMO with Anammox for sustainable wastewater treatment.

SSThe coexistence and diversity of Candidatus methylomirabilis oxyfera-like and anammox bacteria in sediments of an urban eutrophic lake.

Tangxun Lake is the largest urban lake in China, which is polluted by multiple wastewaters, and now is severely eutrophic. We detected diversity, abundance, and the coexistence of Candidatus Methylomirabilis oxyfera-like and anammox bacteria in different horizontal and vertical directions of the lake sediments through qPCR and clone library. Phylogenetic tree analysis showed that the Ca. Methylomirabilis oxyfera-like and anammox bacteria exhibited high diversity, and they belonged to group B-E and Ca. Brocadia genus, respectively. These two bacteria displayed higher diversity in polluted area than in other areas. Furthermore, they had great spatial variation of abundance both horizontally and vertically. The abundance of anammox bacteria was significantly higher than that of Ca. Methylomirabilis oxyfera-like bacteria. The stronger the human interference were, the higher abundances these two bacteria exhibited horizontally, whereas both their abundances and the ratio of anammox to Ca. Methylomirabilis oxyfera-like bacteria decreased with the increasing depth. Redundancy analysis indicated that nitrate was the most influential environmental factor to the abundance of these two bacteria. Ammonia, nitrite, total nitrogen, and organic matters were in positive correlation with the abundance of these two bacteria. Nitrate was slightly negatively correlated with the abundance of Ca. Methylomirabilis oxyfera-like bacteria, while it was positively correlated with that of anammox bacteria. Our results provided an insight into the effects of environmental factors such as ammonia, nitrite, and nitrate on the diversity and abundances of these two bacteria and theoretical basis for restoration of water.

Enhancing mainstream nitrogen removal by employing nitrate/nitrite-dependent anaerobic methane oxidation processes.

Due to serious eutrophication in water bodies, nitrogen removal has become a critical stage for wastewater treatment plants (WWTPs) over past decades. Conventional biological nitrogen removal processes are based on nitrification and denitrification (N/DN), and are suffering from several major drawbacks, including substantial aeration consumption, high fugitive greenhouse gas emissions, a requirement for external carbon sources, excessive sludge production and low energy recovery efficiency, and thus unable to satisfy the escalating public needs. Recently, the discovery of anaerobic ammonium oxidation (anammox) bacteria has promoted an update of conventional N/DN-based processes to autotrophic nitrogen removal. However, the application of anammox to treat domestic wastewater has been hindered mainly by unsatisfactory effluent quality with nitrogen removal efficiency below 80%. The discovery of nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) during the last decade has provided new opportunities to remove this barrier and to achieve a robust system with high-level nitrogen removal from municipal wastewater, by utilizing methane as an alternative carbon source. In the present review, opportunities and challenges for nitrate/nitrite-dependent anaerobic methane oxidation are discussed. Particularly, the prospective technologies driven by the cooperation of anammox and n-DAMO microorganisms are put forward based on previous experimental and modeling studies. Finally, a novel WWTP system acting as an energy exporter is delineated.

Molecular and stable isotopic evidence for the occurrence of nitrite-dependent anaerobic methane-oxidizing bacteria in the mangrove sediment of Zhangjiang Estuary, China.

Nitrite-dependent anaerobic methane oxidation (n-damo), which is mediated by "Candidatus Methylomirabilis oxyfera-like" bacteria, is unique in linking the carbon and nitrogen cycles. However, the niche and activity of n-damo bacteria in the mangrove ecosystem have not been confirmed. Here, we report the occurrence of the n-damo process in the mangrove wetland of the Zhangjiang Estuary, China. The widespread occurrence of n-damo bacteria in mangrove wetland was confirmed using real-time quantitative polymerase chain reaction (qPCR) assay, which showed that the abundance of Methylomirabilis oxyfera-like bacterial 16S rRNA and pmoA genes ranged from 2.43 × 106 to 2.09 × 107 and 2.07 × 106 to 3.38 × 107copies per gram of dry soil in the examined sediment cores. The highest amount of targeting genes was all detected in the upper layer (0-20 cm). Phylogenetic analyses of n-damo bacterial 16S rRNA and pmoA genes illustrated the depth-specific distribution and high diversity of n-damo bacteria in the mangrove wetland. Stable isotope experiments further confirmed the occurrence of n-damo in the examined mangrove sediments, and the potential n-damo rates ranged from 25.93 to 704.08 nmol CO2 per gram of dry soil per day at different depths of the sediment cores, with the n-damo being more active in the upper layer of the mangrove sediments. These results illustrate the existence of active M. oxyfera-like bacteria and indicate that the n-damo process is a previously overlooked microbial methane sink in the mangrove wetlands.

Spatial and temporal distribution of nitrite-dependent anaerobic methane-oxidizing bacteria in an intertidal zone of the East China Sea.

Nitrite-dependent anaerobic methane oxidation (N-DAMO), which couples anaerobic methane oxidation and nitrite reduction, is a recently discovered bioprocess coupling microbial nitrogen and carbon cycles. The discovery of this microbial process challenges the traditional knowledge of global methane sinks and nitrogen losses. In this study, the abundance and activity of N-DAMO bacteria were investigated and their contributions to methane sink and nitrogen loss were estimated in different seasons and different partitions of an intertidal zone of the East China Sea. The results showed that N-DAMO bacteria were extensively and continuously present in the intertidal zone, with the number of cells ranging from 5.5 × 104 to 2.8 × 105 copy g-1 soil and the potential activity ranging from 0.52 to 5.7 nmol CO2 g-1 soil day-1, contributing 5.0-36.6% of nitrite- and sulfate-dependent anaerobic methane oxidation in the intertidal zone. The N-DAMO activity and its contribution to the methane consumption were highest in the spring and in the low intertidal zone. These findings showed that the N-DAMO process is an important methane and nitrogen sink in the intertidal zone and varies with the seasons and the partitions of the intertidal zone.

Improved PCR primers to amplify 16S rRNA genes from NC10 bacteria.

Anaerobic oxidation of methane (AOM) coupled to nitrite reduction (AOM-NIR) is ecologically significant for mitigating the methane-induced greenhouse effect. The microbes responsible for this reaction, NC10 bacteria, have been widely detected in diverse ecosystems. However, some defects were discovered in the commonly used NC10-specific primers, 202F and qP1F. In the present work, the primers were redesigned and improved to overcome the defects found in the previous primers. A new nested PCR method was developed using the improved primers to amplify 16S ribosomal RNA (rRNA) genes from NC10 bacteria. In the new nested PCR method, the qP1mF/1492R and 1051F/qP2R primer sets were used in the first and second rounds, respectively. The PCR products were sequenced, and more operational taxonomic units (OTUs) of the NC10 phylum were obtained using the new primers compared to the previous primers. The sensitivity of the new nested PCR was tested by the serial dilution method, and the limit of detection was approximately 10(3) copies g(-1) dry sed. for the environmental samples compared to approximately 10(5) copies g(-1) dry sed. by the previous method. Finally, the improved primer, qP1mF, was used in quantitative PCR (qPCR) to determine the abundance of NC10 bacteria, and the results agreed well with the activity of AOM-NIR measured by isotope tracer experiments. The improved primers are able to amplify NC10 16S rRNA genes more efficiently than the previous primers and useful to explore the microbial community of the NC10 phylum in different systems.

Impact of hydrogen sulfide on anammox and nitrate/nitrite-dependent anaerobic methane oxidation coupled technologies.

The coupling between anammox and nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) has been considered a sustainable technology for nitrogen removal from sidestream wastewater and can be implemented in both membrane biofilm reactor (MBfR) and granular bioreactor. However, the potential influence of the accompanying hydrogen sulfide (H2S) in the anaerobic digestion (AD)-related methane-containing mixture on anammox/n-DAMO remains unknown. To fill this gap, this work first constructed a model incorporating the C/N/S-related bioprocesses and evaluated/calibrated/validated the model using experimental data. The model was then used to explore the impact of H2S on the MBfR and granular bioreactor designed to perform anammox/n-DAMO at practical levels (i.e., 0∼5% (v/v) and 0∼40 g/S m3, respectively). The simulation results indicated that H2S in inflow gas did not significantly affect the total nitrogen (TN) removal of the MBfR under all operational conditions studied in this work, thus lifting the concern about applying AD-produced biogas to power up anammox/n-DAMO in the MBfR. However, the presence of H2S in the influent would either compromise the treatment performance of the granular bioreactor at a relatively high influent NH4+-N/NO2--N ratio (e.g., >1.0) or lead to increased energy demand associated with TN removal at a relatively low influent NH4+-N/NO2--N ratio (e.g., <0.7). Such a negative effect of the influent H2S could not be attenuated by regulating the hydraulic residence time and should therefore be avoided when applying the granular bioreactor to perform anammox/n-DAMO in practice.

Impacts of granular sludge properties on the bioreactor performing nitrate/nitrite-dependent anaerobic methane oxidation/anammox processes.

In this work, a bioprocess model was applied to first determine the impacts of influent substrates conditions on the granular bioreactor performing nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) and anammox integrated processes and then investigate the roles of granular sludge properties in regulating the bioreactor performance and start-up process. The ideal influent substrates conditions were identified at NO2--N/NH4+-N of 1:1 and dissolved CH4 concentration of 85 g COD m-3, which achieved 98.6% total nitrogen removal and 87.7% dissolved CH4 utilization. Under such ideal influent conditions, the initial properties of granular sludge didn't significantly affect the granular bioreactor performance. However, inoculation of granular sludge with a relatively small granular sludge size and a high abundance of n-DAMO archaea or/and anammox bacteria could effectively shorten the bioreactor start-up. Meanwhile, reducing the diffusivity of solutes within granular sludge was also beneficial for expediting the start-up process and promoting dissolved CH4 utilization.

Instrumental role of bioreactors in nitrate/nitrite-dependent anaerobic methane oxidation-based biotechnologies for wastewater treatment: A review.

Recently, the nitrate/nitrite-dependent anaerobic methane oxidation (n-DAMO) processes have become a research hotspot in the field of wastewater treatment. The n-DAMO processes could not only mitigate direct and indirect carbon emissions from wastewater treatment plants but also strengthen biological nitrogen removal. However, the applications of n-DAMO-based biotechnologies face practical difficulties mainly caused by the distinctive properties of n-DAMO microorganisms and the limited/availability of methane with poor solubility. In this sense, the choice of bioreactors will play important roles that influence the growth and functioning of n-DAMO microorganisms, thus enabling dedicated development of the n-DAMO processes and efficient applications of n-DAMO-based biotechnologies. Therefore, this paper aims to discuss the three commonly-applied types of bioreactors, covering the individual working principle and state-of-the-art removal performance of nitrogen as well as dissolved methane observed when adopted for n-DAMO-based biotechnologies. With noted limitations for each bioreactor type, several key perspectives were proposed which hopefully would inspire future investigation and practical applications of the n-DAMO processes.

Making good use of methane to remove oxidized contaminants from wastewater.

Being an energetic fuel, methane is able to support microbial growth and drive the reduction of various electron acceptors. These acceptors include a broad range of oxidized contaminants (e.g., nitrate, nitrite, perchlorate, bromate, selenate, chromate, antimonate and vanadate) that are ubiquitously detected in water environments and pose threats to human and ecological health. Using methane as electron donor to biologically reduce these contaminants into nontoxic forms is a promising solution to remediate polluted water, considering that methane is a widely available and inexpensive electron donor. The understanding of methane-based biological reduction processes and the responsible microorganisms has grown in the past decade. This review summarizes the fundamentals of metabolic pathways and microorganisms mediating microbial methane oxidation. Experimental demonstrations of methane as an electron donor to remove oxidized contaminants are summarized, compared, and evaluated. Finally, the review identifies opportunities and unsolved questions that deserve future explorations for broadening understanding of methane oxidation and promoting its practical applications.

Achieving high nitrogen removal efficiency by optimizing nitrite-dependent anaerobic methane oxidation process with growth factors.

Nitrite-dependent anaerobic methane oxidation (N-DAMO) is a newly discovered bioprocess which uses methane as electron donor to reduce nitrite into dinitrogen. It is a promising clean bioprocess for denitrification in wastewater treatment. However, the low reaction rate and slow growth rate of N-DAMO bacteria within NC10 phylum limit the application of the process. In this study, we chose vitamin, heme, nucleobase and betaine to investigate their short- and long-term effects on N-DAMO bacteria. The concentrations of the growth factors of medium were improved according to the short-term experiments. The results were subsequently verified via long-term inoculations and were applied in a magnetically stirred gas lift reactor (MSGLR). The results indicated that nucleobase and betaine (5.0 and 200 µg L-1, respectively) significantly stimulated the N-DAMO activity, whereas vitamin and heme had no significant effects in the tested concentration ranges. During the long-term incubation, N-DAMO bacteria continuously increased and finally achieved a relative abundance of 14.4% on day 300. Notably, larger aggregates of N-DAMO bacteria were observed at the end of the long-term incubation. And the nitrogen removal rate of the MSGLR increased to 70 mg N L-1 day-1, with the total nitrogen removal efficiency over 99.0%. However, the addition of betaine introduced methyl into the reactors and this made methylotrophs account a considerable part of the bacterial community, which limited the enrichment degree of N-DAMO bacteria. This work will contribute to the engineering application and enrichment of N-DAMO bacteria.

[Rapid Start-up of a Nitrite-Dependent Methane Anaerobic Oxidation Reaction Under Static Pressure Conditions].

The present study explores the effect of static pressure on the rapid start-up of a nitrite-dependent anaerobic methane oxidation (N-DAMO) process in lab-scale sequencing batch reactors (SBR). A mixture of anaerobic sludge and deep paddy soil with a volume ratio of 1:1 was used as inoculum and the influent of the nitrite (NO2--N) concentration was gradually increased to avoid a toxicity shock. The variation of the NO2--N removal performance and corresponding microbial characteristics were analyzed to evaluate the development of the N-DAMO process. After 120 days of operation, significant N-DAMO phenomena were observed in both the control SBR (R1) with normal pressure and pressurized SBR (R2) with a static pressure of 0.3 MPa. The NO2--N removal rate (measured by NO2--N) of R2 (36.90 mg·(L·d)-1) was 24% higher than that of R1, while the average NO2--N removal rate in the first 4 h of the batch cycle in R2 (0.10 mmol·(L·h)-1) was 186% higher than that of R1. The mean sludge size of R2 was~2-fold larger than that of R1. Sludge in R2 also has a bigger specific surface area, which improves the mass transfer rate of methane and the N-DAMO performance. The specific activity of N-DAMO (measured by N/VSS) reached 0.29 mg·(g·h)-1 in the study period, which is approximately 2 times higher than that of R1. Moreover, the abundance of N-DAMO functional microbes Candidatus Methylomirabilish oxyfera (M. oxyfera) in R2 was 10-fold higher than that of R1. These results indicate that static pressure effectively accelerates the start-up of the N-DAMO process.

Effect of trace elements on the development of co-cultured nitrite-dependent anaerobic methane oxidation and methanogenic bacteria consortium.

The aim of this work was to study the effects of key trace elements (i.e., iron, copper and molybdenum) on the development of co-cultured n-damo and methanogenic bacteria consortium, which could realize in situ CH4 production and utilization. The results showed that rational dosage, which was 50 mg/L of Fe, 1 mg/L of Cu and 5 mg/L of Mo, significantly stimulated the removal of NO2-. However, the activity of microbes was noticeably inhibited at 5 mg/L of Cu and 1 mg/L of Mo. Microbial community analysis indicated that the abundances of n-damo bacteria and methanogens showed a positive response to the rational dosage. Furthermore, the expression of key functional genes was enhanced under the rational dosage condition.

Improvement of the trace metal composition of medium for nitrite-dependent anaerobic methane oxidation bacteria: Iron (II) and copper (II) make a difference.

Nitrite-dependent anaerobic methane oxidation (n-damo) is a potential bioprocess for treating nitrogen-containing wastewater. This process uses methane, an inexpensive and nontoxic end-product of anaerobic digestion, as an external electron donor. However, the low turnover rate and slow growth rate of n-damo functional bacteria limit the practical application of this process. In the present study, the short- and long-term effects of variations in trace metal concentrations on n-damo bacteria were investigated, and the concentrations of trace metal elements of medium were improved. The results were subsequently verified by a group of long-term inoculations (90 days) and were applied in a sequencing batch reactor (SBR) (84 days). The results indicated that iron (Fe(II)) and copper (Cu(II)) (20 and 10 µmol L(-1), respectively) significantly stimulated the activity and the growth of n-damo bacteria, whereas other trace metal elements, including zinc (Zn), molybdenum (Mo), cobalt (Co), manganese (Mn), and nickel (Ni), had no significant effect on n-damo bacteria in the tested concentration ranges. Interestingly, fluorescence in situ hybridization (FISH) showed that a large number of dense, large aggregates (10-50 µm) of n-damo bacteria were formed by cell adhesion in the SBR reactor after using the improved medium, and to our knowledge this is the first discovery of large aggregates of n-damo bacteria.

SSThe coexistence and diversity of Candidatus methylomirabilis oxyfera-like and anammox bacteria in sediments of an urban eutrophic lake / SLa coexistencia y diversidad de bacterias Candidatus methylomirabilis oxyfera y anammox en sedimentos de un lago urbano eutrófico

Tangxun Lake is the largest urban lake in China, which is polluted by multiple wastewaters, and now is severely eutrophic. We detected diversity, abundance, and the coexistence of Candidatus Methylomirabilis oxyfera-like and anammox bacteria in different horizontal and vertical directions of the lake sediments through qPCR and clone library. Phylogenetic tree analysis showed that the Ca. Methylomirabilis oxyfera-like and anammox bacteria exhibited high diversity, and they belonged to group B—E and Ca. Brocadia genus, respectively. These two bacteria displayed higher diversity in polluted area than in other areas. Furthermore, they had great spatial variation of abundance both horizontally and vertically. The abundance of anammox bacteria was significantly higher than that of Ca. Methylomirabilis oxyfera-like bacteria. The stronger the human interference were, the higher abundances these two bacteria exhibited horizontally, whereas both their abundances and the ratio of anammox to Ca. Methylomirabilis oxyfera-like bacteria decreased with the increasing depth. Redundancy analysis indicated that nitrate was the most influential environmental factor to the abundance of these two bacteria. Ammonia, nitrite, total nitrogen, and organic matters were in positive correlation with the abundance of these two bacteria. Nitrate was slightly negatively correlated with the abundance of Ca. Methylomirabilis oxyfera-like bacteria, while it was positively correlated with that of anammox bacteria. Our results provided an insight into the effects of environmental factors such as ammonia, nitrite, and nitrate on the diversity and abundances of these two bacteria and theoretical basis for restoration of water.(AU)