Bioaugmentation of intertidal sludge enhancing the development of salt-tolerant aerobic granular sludge.

Three parallel bioreactors were operated with different inoculation of activated sludge (R1), intertidal sludge (ItS) (R2), and ItS-added AS (R3), respectively, to explore the effects of ItS bioaugmentation on the formation of salt-tolerant aerobic granular sludge (SAGS) and the enhancement of COD removal performance. The results showed that compared to the control (R1-2), R3 promoted a more rapid development of SAGS with a cultivation time of 25 d. Following 110-day cultivation, R3 exhibited a higher granular diameter of 1.3 mm and a higher hydrophobic aromatic protein content than that in control. Compared to the control, the salt-tolerant performance in R3 was also enhanced with the COD removal efficiency of 96.4% due to the higher sludge specific activity of 14.4 g·gVSS-1·d-1 and the salinity inhibition constant of 49.3 gL-1. Read- and genome-resolved metagenomics together indicated that a higher level of tryptophan/tyrosine synthase gene (trpBD, tyrBC) and enrichment of the key gene hosts Rhodobacteraceae, Marinicella in R3, which was about 5.4-fold and 1.4-fold of that in control, could be the driving factors of rapid development of SAGS. Furthermore, the augmented salt-tolerant potential in R3 could result from that R1 was dominated by Rhodospirillaceae, Bacteroidales, which carried more trehalose synthase gene (otsB, treS), while the dominant members Rhodobacteraceae, Marinicella in R3 were main contributors to the glycine betaine synthase gene (ectC, betB, gbsA). This study could provide deeper insights into the rapid development and improved salt-tolerant potential of SAGS via bioaugmentation of intertidal sludge, which could promote the application of hypersaline wastewater treatment.

The effect of quorum sensing on performance of salt-tolerance aerobic granular sludge: linking extracellular polymeric substances and microbial community.

Aerobic granular sludge (AGS) technology is generally negatively affected by the salinity in high saline organic wastewater. The effect of salinity on organic pollutants removal of AGS was studied in three parallel sequencing batch reactors. The results indicated that the performance of reactors operating at relative low salinity (1%) remained stable. However, at medium salinity (2%) and higher salinity (4%) conditions, the organic pollutants removal efficiencies deteriorated from 93.7 ± 3.0 to 71.6 ± 6.8 and 53.6 ± 5.4%, respectively. The addition of a mixture of acyl-homoserine lactone (AHL) mediated quorum sensing (QS) signaling molecules (0.1 µmol/L of mixed C6-HSL, C8-HSL and 3OC8-HSL) only restored the performance of the 2% salinity reactor back to 86.3 ± 6.2% due to the changing of hydrophobic extracellular polymeric substance ratio from 64 ± 3 to 71 ± 4%. Addition of the AHL had no effect on the pollution removal efficiency at the 4% salinity conditions. Microbial community analysis showed that Dyella (32.3%) species were the dominant member of the community and its occurrence was positively correlated with organic pollutants removal efficiency at relative high salinity (2% and 4%), while Mangrovibacter showed the opposite trend. Higher abundances of hdtS and acylase genes, the synthesis and degradation genes of AHL, were found after adding AHLs to reactors at 2% salinity, which indicated that AHL mediated QS was the primary QS system in salt-tolerant AGS.

Salinity-Aided Selection of Progressive Onset Denitrifiers as a Means of Providing Nitrite for Anammox.

Anaerobic ammonium oxidation (anammox) combined with partial-denitrification (NO3- → NO2-) is an innovative process for the simultaneous removal of ammonia and nitrate from wastewaters. An efficient method for the selection of partial denitrifying community, which relies on increasing influent salinity, is described. Using this method, a denitratating community was enriched, which showed a nitrite accumulation efficiency higher than 75% as well as a high nitrate conversion efficiency. Community analysis using 16S rDNA indicated that Halomonas became the dominant genus as salinity increased. Metagenomic analysis revealed that there was not a significant difference in reads mapping to downstream denitrification genes in a comparison of samples from cultures with 5% salinity to those without salinity. The majority of the reads mapping to the genes encoding dissimilatory nitrate and nitrite reductases nar and nirS came from Halomonas under high salinity conditions. Two metagenome-assembled genomes taxonomically assigned to Halomonas were obtained, one of which accounted for ∼35% of the reads under high salinity conditions. Both genomes harbored the genes for the complete denitrification pathway. These results indicate progressive onset denitrifiers, a phenotype where nitrite reduction only occurs after nitrate exhaustion, could be successfully enriched with increasing salinity. Progressive onset denitrifiers may be more widespread in natural and artificial habitats than anticipated and are shown here to be valuable for nitrogen mitigating processes.

High-throughput screening for a moderately halophilic phenol-degrading strain and its salt tolerance response.

A high-throughput screening system for moderately halophilic phenol-degrading bacteria from various habitats was developed to replace the conventional strain screening owing to its high efficiency. Bacterial enrichments were cultivated in 48 deep well microplates instead of shake flasks or tubes. Measurement of phenol concentrations was performed in 96-well microplates instead of using the conventional spectrophotometric method or high-performance liquid chromatography (HPLC). The high-throughput screening system was used to cultivate forty-three bacterial enrichments and gained a halophilic bacterial community E3 with the best phenol-degrading capability. Halomonas sp. strain 4-5 was isolated from the E3 community. Strain 4-5 was able to degrade more than 94% of the phenol (500 mg · L(-1) starting concentration) over a range of 3%-10% NaCl. Additionally, the strain accumulated the compatible solute, ectoine, with increasing salt concentrations. PCR detection of the functional genes suggested that the largest subunit of multicomponent phenol hydroxylase (LmPH) and catechol 1,2-dioxygenase (C12O) were active in the phenol degradation process.

Aerobic biodegradation of trichloroethylene and phenol co-contaminants in groundwater by a bacterial community using hydrogen peroxide as the sole oxygen source.

Trichloroethylene (TCE) and phenol were often found together as co-contaminants in the groundwater of industrial contaminated sites. An effective method to remove TCE was aerobic biodegradation by co-metabolism using phenol as growth substrates. However, the aerobic biodegradation process was easily limited by low concentration of dissolved oxygen (DO) in groundwater, and DO was improved by air blast technique with difficulty. This study enriched a bacterial community using hydrogen peroxide (H2O2) as the sole oxygen source to aerobically degrade TCE by co-metabolism with phenol in groundwater. The enriched cultures were acclimatized to 2-8 mM H2O2 which induced catalase, superoxide dismutase and peroxidase to decompose H2O2 to release O2 and reduce the toxicity. The bacterial community could degrade 120 mg/L TCE within 12 days by using 8 mM H2O2 as the optimum concentration, and the TCE degradation efficiency reached up to 80.6%. 16S rRNA gene cloning and sequencing showed that Bordetella, Stenotrophomonas sp., Sinorhizobium sp., Variovorax sp. and Sphingobium sp. were the dominant species in the enrichments, which were clustered in three phyla: Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. Polymerase chain reaction detection proved that phenol hydroxylase (Lph) gene was involved in the co-metabolic degradation of phenol and TCE, which indicated that hydroxylase might catalyse the epoxidation of TCE to form the unstable molecule TCE-epoxide. The findings are significant for understanding the mechanism of biodegradation of TCE and phenol co-contamination and helpful for the potential applications of an aerobic bioremediation in situ the contaminated sites.

Effects of superficial gas velocity on the performance of an air-lift internal circulation partial nitrification-anammox granular sludge reactor.

The airlift internal circulation reactor for partial nitrification-anammox (PNA-ALR) has the advantages of a small footprint, high mass transfer efficiency, and the ease of formation of granular sludge, thus making it an effective biological treatment for ammonia-containing wastewater. Although superficial gas velocity (SGV) is an essential parameter for PNA-ALR, it is unclear how the magnitude of SGV impacts nitrogen removal performance. In this study, the nitrogen removal efficiencies of five PNA-ALRs with different SGV were measured during feeding with synthetic municipal wastewater. At an optimal SGV of 2.35 cm s-1, the PNA-ALR consistently maintained the total inorganic nitrogen (TIN) removal efficiency at 76.31% and the effluent TIN concentration was less than 10 mg L-1. By increasing or decreasing the SGV, the nitrogen removal efficiency decreased to a range between 30% and 50%. At lower SGV, the dead space in the PNA-ALR was increased by 21.15%, and the feast/famine ratio of sludge increased to greater than 0.5, which caused a disruption in the structure, and a large loss of, granular sludge. Computational fluid dynamics (CFD) simulations showed operation at a higher SGV, resulting in excessive shear stress of 3.25 N m-2 being generated from bubble rupture in the degassing section. Fluorescent staining determined a decrease of 26.5% in viable bacteria. These results have improved our understanding of the effects of SGV on a PNA-ALR during mainstream wastewater treatment.

Effects of 1,1,1-trichloroethane on enzymatic activity and bacterial community in anaerobic microcosm form sequencing batch reactors.

1,1,1-Trichloroethane (TCA), a major organic and groundwater contaminant, has very strong toxic effects on humans, plants and microorganisms. Effects of TCA on enzymatic activity and microbial diversity were investigated in the anaerobic sequencing batch reactor (ASBR) under methanogenic, nitrate-reducing, sulfate-reducing and benzene/toluene degrading conditions. The activities of three enzymes (lactate dehydrogenase, phosphatase and protease) were significantly decreased in the presence of 5 mg/L TCA. Within these three affected enzymes, phosphatase activity may serve as a noteworthy marker of bacterial toxicity. The activity of phosphatase was 0.2 U/L in methanogenic conditions with 5 mg/L TCA, which was 99% lower than the controls, and the enzyme activity was 18.6 U/L in methanogenic conditions with 1 mg/L TCA, which was 7% lower than the controls. DGGE profiles showed that TCA altered the bacterial community distribution and diversity obviously during the 21 day of TCA exposure. The enzyme activities decreased second lowest but TCA degrading strains Clostridium sp. DhR-2/LM-G01, Bacterial clone DCE25 and Bacterial clone DPHB06 were enriched in the methanogenic ASBR amended TCA.

Saline short-term shock and rapid recovery on anammox performance.

Anaerobic ammonia oxidation (anammox) is an environmental-friendly biological nitrogen removal process, which has been developed as a promising technology in industrial wastewater treatment. However, anammox nitrogen removal under high saline conditions still faces many challenges. This study investigated the performance of anammox sludge under saline short-term shock and the strategy of rapid recovery. Salinity concentration, saline exposure time, and NaCl/Na2SO4 ratio were selected as three critical factors for short-term shock. The activity inhibition of anammox sludge were tested by using response surface methodology (RSM). Our results showed that, compared with the NaCl/Na2SO4 ratio, the salinity concentration and saline exposure time were the significant factor causing the anammox inhibition. The addition of glycine betaine (GB) in moderate amounts (0.1-5 mM) was found to help anammox to resist in relative low saline shock intensities (e.g., IC25 and IC50), with the activity retention rate of 94.7%. However, glycine betaine was not worked effectively under relatively high saline shock intensities (e.g., complete inhibition condition). Microbial community analysis revealed that Brocadiaceae accounted for only about 7.6%-13.2% at inhibited conditions. Interestingly, 16S rRNA analysis showed that the abundance of activated Brocadiaceae remarkably decreased with time after high-level saline shock. This tendency was consistent with the results of qPCR targeted hzsA gene. Finally, based on quorum sensing, the anammox activity was recovered to 93.5% of original sludge by adding 30% original sludge. The study realized the rapid recovery of anammox activity under complete inhibition, promoting the development and operation of salt-tolerant anammox process.

[Metagenomic and Metatranscriptomic Analysis of Nitrogen Removal Functional Microbial Community of Petrochemical Wastewater Biological Treatment Systems].

Petrochemicals are one of the pillar industries of China. Despite this, the treatment of petrochemical wastewater has long been seen as a massive challenge in the field of water pollution control, hindering the high-quality and sustainable development of the petrochemical industry. The majority of petrochemical enterprises and zones are located near rivers or seas, so their wastewater discharges can easily cause watershed or regional water ecological risks. Specifically, nitrogen pollution in petrochemical wastewater poses a significant threat to water ecological safety and human health. Sludge samples were collected from a petrochemical wastewater A/O nitrogen removal process line in a chemical industry zone in Shanghai. Metagenomic and metatranscriptomic methods were used to analyze the community structure of microorganisms, the functional characteristics of nitrogen removal bacteria, and the key nitrogen metabolism pathways in different sludges during the period when effluent water quality was stable and fluctuating. During the study, it was found that the nitrite and nitrate removal was relatively stable in this process, but ammonia oxidation fluctuated easily. In the study of microbial communities, it was found to be a nitrification-denitrification pathway that primarily removed nitrogen from the A/O process, and no genes related to ANAMMOX were detected. Approximately 90% of the functional genes responsible for removing nitrogen were responsible for denitrification, whereas only 0.17% of them were involved in the conversion of ammonia nitrogen in the nitrification process. Moreover, the abundance of ammonia-oxidizing bacteria in the process was extremely low, and the main genus was Nitrosomonas. It is likely that this is the main cause of fluctuations in ammonia nitrogen concentration in effluent due to water quality shocks in the process line.

A novel hydrocyclone for use in underground DNAPL phase separation.

Halogenated organic solvents are the most commonly detected pollutants in groundwater and are particularly toxic and harmful. How to separate these dense nonaqueous phase liquid (DNAPL) pollutants efficiently from groundwater has become an important research question. Here, a novel hydrocyclone with annular overflow structure was designed, which eliminated the short-circuit flow of the traditional hydrocyclone and solved the problem of overflow entrainment caused by the enrichment of droplets near the locus of zero vertical velocities (LZVV) into turbulence. The flow field characteristics of this novel hydrocyclone were studied using Computational Fluid Dynamics (CFD) simulation and compared with the traditional hydrocyclone. It was found that the annular gap structure of the novel hydrocyclone increased the tangential velocity of the outer vortex. Moreover, the radius of the LZVV was expanded outward by 0.17 mm, which reduced the possibility of droplets with small particle sizes in the second phase escaping from the overflow pipe. The collective effect was to eliminate the short-circuit flow. This novel hydrocyclone was able to separate DNAPL pollutants with low consumption and high efficiency, across a range of inlet velocity from 4 to 6 m/s. The maximum separation efficiency was 99.91 %. In addition, with trichloroethylene (TCE) as the target pollutant, the maximum volume fraction of the dispersed phase in the hydrocyclone was located on the side wall of the hydrocyclone. Taken together, we believe that this work will provide a low-cost, efficient separation method for the separation of groundwater- contaminated liquid mixtures. Furthermore, it has broad application prospects in the field of heterotopic remediation of groundwater.

Halophilic Martelella sp. AD-3 enhanced phenanthrene degradation in a bioaugmented activated sludge system through syntrophic interaction.

Polycyclic aromatic hydrocarbons (PAHs) are a group of common recalcitrant pollutant in industrial saline wastewater that raised significant concerns, whereas traditional activated sludge (AS) has limited tolerance to high salinity and PAHs toxicity, restricting its capacity to degrade PAHs. It is therefore urgent to develop a bioaugmented sludge (BS) system to aid in the effective degradation of these types of compounds under saline condition. In this study, a novel bioaugmentation strategy was developed by using halophilic Martelella sp. AD-3 for effectively augmented phenanthrene (PHE) degradation under 3% salinity. It was found that a 0.5∼1.5% (w/w) ratio of strain AD-3 to activated sludge was optimal for achieving high PHE degradation activity of the BS system with degradation rates reaching 2.2 mg⋅gVSS-1⋅h-1, nearly 25 times that of the AS system. Although 1-hydroxy-2-naphthoic acid (1H2N) was accumulated obviously, the mineralization of PHE was more complete in the BS system. Reads-based metagenomic coupled metatranscriptomic analysis revealed that the expression values of ndoB, encoding a dioxygenase associated with PHE ring-cleavage, was 5600-fold higher in the BS system than in the AS system. Metagenome assembly showed the members of the Corynebacterium and Alcaligenes genera were abundant in the strain AD-3 bioaugmented BS system with expression of 10.3±1.8% and 1.9±0.26%, respectively. Moreover, phdI and nahG accused for metabolism of 1H2N have been annotated in both above two genera. Degradation assays of intermediates of PHE confirmed that the activated sludge actually possessed considerable degradation capacity for downstream intermediates of PHE including 1H2N. The degradation capacity ratio of 1H2N to PHE was 87% in BS system, while it was 26% in strain AD-3. These results indicated that strain AD-3 contributed mainly in transforming PHE to 1H2N in BS system, while species in activated sludge utilized 1H2N as substrate to grow, thus establishing a syntrophic interaction with strain AD-3 and achieving the complete mineralization of PHE. Long-term continuous experiment confirmed a stable PHE removal efficiency of 93% and few 1H2N accumulation in BS SBR system. This study demonstrated an effective bioaugmented strategy for the bioremediation of saline wastewater containing PAHs.

The anammox coupled partial-denitrification process in an integrated granular sludge and fixed-biofilm reactor developed for mainstream wastewater treatment: Performance and community structure.

This study describes an integrated granular sludge and fixed-biofilm (iGB) reactor innovatively designed to carry out the anammox/partial-denitrification (A/PD) process for nitrogen removal with mainstream municipal wastewater. The iGB-A/PD reactor consists of anammox granules inoculated in the lower region of reactor and an acclimated fixed-biofilm positioned in the upper region. Compared to the other reported A/PD systems for mainstream wastewater treatment, this iGB-A/PD reactor is notable due to its higher quality effluent with a total inorganic nitrogen (TIN) of ∼3 mg•L-1 and operation at a high nitrogen removal rate (NRR) of 0.8 ± 0.1 kg-N•m-3•d-1. Reads-based metatranscriptomic analysis found that the expression values of hzsA and hdh, key genes associated with anammox, were much higher than other functional genes on nitrogen conversion, confirming the major roles of the anammox bacteria in nitrogen bio-removal. In both regions of the reactor, the nitrate reduction genes (napA/narG) had expression values of 56-99 RPM, which were similar to that of the nitrite reduction genes (nirS/nirK). The expression reads from genes for dissimilatory nitrate reduction to ammonium (DNRA), nrfA and nirB, were unexpectedly high, and were over the half of the levels of reads from genes required for nitrate reduction. Kinetic assays confirmed that the granules had an anammox activity of 16.2 g-NH4+-N•kg-1-VSS•d-1 and a nitrate reduction activity of 4.1 g-N•kg-1-VSS•d-1. While these values were changed to be 4.9 g- NH4+-N•kg-1-VSS•d-1and 4.3 g-N•kg-1-VSS•d-1 respectively in the fixed-biofilm. Mass flux determination found that PD and DNRA was responsible for ∼50% and ∼25% of nitrate reduction, respectively, in the whole reactor, consistent with high effluent quality and treatment efficiency via a nitrite loop. Metagenomic binning analysis revealed that new and unidentified anammox species, affiliated with Candidatus Brocadia, were the dominant anammox organisms. Myxococcota and Planctomycetota were the principal organisms associated with the PD and DNRA processes, respectively.

[Cultivation and Performance Analysis of Simultaneous Partial Nitrification, ANAMMOX, and Denitratation Granular Sludge].

We cultivated simultaneous partial nitrification, anaerobic ammonium oxidizing(ANAMMOX), and denitratation granular sludge in a novel air-lift internal circulation reactor using low C/N wastewater as the substrate and ANAMMOX sludge matched with ordinary activated sludge as the inoculum. The results showed that the mature and stable granular sludge could be cultivated after 225 d of continuous operation, and the total nitrogen removal rate was as high as 91.4%. Compared with flocculated sludge, the ANAMMOX activity in the granular sludge increased significantly, and the ANAMMOX activity was highest among the four nitrogen removal processes followed by partial nitrification, and the specific denitratation activity was 2.1-times higher than the specific nitrite reduction activity. High-throughput sequencing results showed that the dominant bacteria in partial nitrification and ANAMMOX were Nitrosomonas and Candidatus_Brocadia, respectively, compared to flocculated sludge, with abundances increasing to 0.70% and 0.57%, respectively. Thauera may also be the potential dominant bacteria for denitratation, with an abundance of up to 0.26%. RT-qPCR analysis showed that compared to the inoculation stage, the transcript levels of the amoA and hao genes for partial nitrification increased 3.5-and 1.5-fold, respectively, and the transcript levels of the hzsA gene for ANAMMOX increased 2.1-fold. During denitrataion, the overall abundance of napA and narG transcript levels was 4.8-times higher than that of nirK and nirS. The results of this study provide new insights for the treatment of low C/N wastewater.

Micro-nano aeration is a promising alternative for achieving high-rate partial nitrification.

Biological nitrogen removal is the most prevalent wastewater nitrogen removal process but nitrification limits the rate of the whole process mainly due to the low efficiency of oxygen transfer. In this study, clean-water oxygenation tests, batch tests, long-term operational tests and metagenomic analyses were applied to assess the effects of micro-nano aeration on nitrification. The oxygen transfer coefficient (KLa), oxygen transfer rate (OTR) and oxygen transfer efficiency (OTE) were determined to be 0.56 min-1, 0.36 kg·m-3·h-1 and 71.43%, respectively during micro-nano-bubble aeration. Impressively, these values were 15 times greater than those of conventional aeration. The results of batch tests and long-term operation experiments found that the ammonia removal rate of micro-nano aeration was 3.2-fold that of conventional aeration. The energy cost for micro-nano aeration was calculated to be 3694.5 mg NH4+-N/kW·h, a 50% energy saving in comparison to conventional aeration. In addition, the nitrite accumulation ratio in the Micro-nano (MN) reactor was 1.5 that of the Conventional (CV) reactor. Metagenomic analysis showed that after long-term operation in micro-nano aeration, the abundances of genes encoding ammonia monooxygenase (amoA) and hydroxylamine oxidoreductase (hao) was more than 8-fold and 4-fold of those in conventional aeration, respectively. The abundance of the gene encoding nitrite oxidoreductase (nxrA) was similar in both reactors. Read taxonomy revealed that abundance of AOB-Nitrosomonas increased significantly when using micro-nano aeration, while abundance of NOB-Nitrospira abundance was similar in both reactors. The results of this study indicated that the micro-nano aeration process will increase the ammonia oxidation performance by enhancing oxygen transfer but was also shown to be beneficial for enhancing partial nitrification by specific enrichment of ammonia oxidizing bacteria. This latter result demonstrates the potential benefits of the micro-nano aeration process as an alternative approach to establishing high-rate partial nitrification.

Metagenomics and metatranscriptomics uncover the microbial community associated with high S0 production in a denitrifying desulfurization granular sludge reactor.

The denitrification desulfurization process is a promising technology for elemental sulfur (S0) production from sulfide containing wastewater. However, the microbial community associated with high S0 production still is not well studied. This study describes an efficient denitrification S0 production bioreactor based on inoculation with anaerobic granular sludge. At an optimal S/N molar ratio of 7:2, 80 % of the influent sulfide was transformed to high quality elemental sulfur with a purity of 92.5% while the total inorganic nitrogen removal efficiency was stable at ∼80%. Metatranscriptomic analysis found that community expression of the gene encoding the sulfide-quinone reductase (SQR) was 10-fold greater than that of the flavocytochrome-c sulfide dehydrogenase subunit B (fccB). Moreover, the expression level of SQR was also significantly higher than the Dsr gene encoding for dissimilatory sulfate reductase, which encodes a critical S0 oxidation enzyme. Metagenomic binning analysis confirmed that sulfide-oxidizing bacteria (SOB) utilizing SQR were common in the community and most likely accounted for high S0 production. An unexpected enrichment in methanogens and high expression activity of bacteria carrying out Stickland fermentation as well as in other bacteria with reduced genomes indicated a complex community supporting stable sulfide oxidation to S0, likely aiding in performance stability. This study establishes this treatment approach as an alternative biotechnology for sulfide containing wastewater treatment and sheds light on the microbial interactions associated with high S0 production.

The short- and long-term effects of formic acid on rapid nitritation start-up.

The feasibility of achieving stable nitritation inoculating with activated sludge by adding formic acid was studied in this work. Short-term batch effects of formic acid on nitrification showed that the nitrite accumulation ratio (NAR) significantly increased from 0.3% to 83.7% with an increase of formic acid concentration from 0 to 50 mM at an initial ammonia concentration of 75 mg·L-1, which was demonstrated to be due to the inhibition of nxrB transcription in nitrite oxidizing bacteria (NOB). The long-term effects of formic acid at 30 mM were constantly monitored in an aerobic sequencing batch reactor. During 27 days of operation, the NAR was rapidly raised and maintained approximately 90%. What's more, in the following 52 days without addition of formic acid, the NAR was kept above 91.3%. The sustained suppression of NOB genus Nitrospira coupling nxrB inhibition was the main reason to maintain stable nitritation. These results supported the feasibility of formic acid as an efficient nitritation regulator, thus providing a new approach for the development of the BNR process via nitrite pathway.

Flocs are the main source of nitrous oxide in a high-rate anammox granular sludge reactor: insights from metagenomics and fed-batch experiments.

Nitrous oxide (N2O) emissions from anammox-based processes are well documented but insight into source of the N2O emission in high-rate anammox granular sludge reactors (AGSR) is limited. In this study, metagenomics and fed-batch experiments were applied to investigate the relative contributions of anammox granules and flocs to N2O production in a high-rate AGSR. Flocs, which constitute only ~10% of total biomass contributed about 60% of the total N2O production. Granules, the main contributor of nitrogen removal (~95%), were responsible for the remaining ~40% of N2O production. This result is inconsistent with reads-based analysis that found the gene encoding clade II type nitrous oxide reductase (nosZII) had similar abundances in both granules and flocs. Another notable trend observed was the relatively higher abundance of the gene for NO-producing nitrite reductase (nir) in comparison to the gene for the nitric oxide reductase gene (nor) in both granules and flocs, indicating nitric oxide (NO) may accumulate in the AGSR. This is significant since NO and N2O pulse assays demonstrated that NO could lead to N2O production from both granules and flocs. However, since anammox bacteria, which were shown to be in higher abundance in granules than in flocs, have the capacity to scavenge NO this provides a mechanism by which its inhibitory effects can be mitigated, limiting N2O release from the granules, consistent with experimental observation. These results demonstrate flocs are the main source of N2O emission in AGSR and provide lab-scale evidence that NO-dependent anammox can mitigate N2O emission.

Application of acidic conditions and inert-gas sparging to achieve high-efficiency nitrous oxide recovery during nitrite denitrification.

Nitrogen removal with energy recovery through denitrification dependent N2O production is garnering recent attention due to its cost advantages. The most effective current method requires alternating COD and nitrite to achieve high N2O production making it incompatible with typical wastewaters and consequently difficult to use in most settings. The work described here introduces a robust and highly efficient N2O recovery approach which has the potential to work with wastewaters containing COD and nitrite simultaneously. This method relies on low pH incubation and inert gas sparging (IGS) to shift a community of mainly N2 producing nitrite denitrifiers to a community that accumulates N2O when incubated in the absence of IGS. Before experiencing IGS, samples from activated sludge incubated at a pH of 4.5 and 6.0 only achieved a maximum N2O production efficiency (PE_N2O) of ∼26%. After IGS the PE_N2O values increased to ∼97.5% and ∼80.2% for samples from these same pH 4.5 and pH 6.0 reactors, respectively. IGS did not lead to N2O production in a pH 7.5 bioreactor. Meta-omics analysis revealed that IGS resulted in an increase in bacteria utilizing the clade I nitrous oxide reductase (nosZI) relative to bacteria utilizing the clade II nitrous oxide reductase (nosZII). This likely results from IGS flushing out N2O leaving nitrite as the principal nitrogen oxide available for respiration, favoring nosZI utilizing bacteria which are more likely to be complete denitrifiers. Metatranscriptomic analysis suggested that the high PE_N2O values that occurred after stopping IGS result from the NO generated by chemodenitrification accumulating to levels that inactivate [4Fe:4S] clusters in the NosR protein essential for N2O reduction in the nosZI denitrifiers. This study provides an efficient and straightforward method for N2O recovery, widening the options for energy recovery from nitrogen-based wastes.

Bacteriophage-mediated extracellular DNA release is important for the structural stability of aerobic granular sludge.

The aim of this study was to investigate the microbial characteristics and the structural role of exDNA in different size AGSs. Metagenomic results showed that exDNA has a significantly lower GC content, ~46.0%, than the ~65.0% GC of intracellular DNA (inDNA). Taxonomic predictions showed most of the reads from the exDNA that could be taxonomically assigned were from members of the phyla Bacteroidetes (55.0-64.2% of the total exDNA reads). Assigned inDNA reads were mainly from Proteobacteria (50.9-57.8%) or Actinobacteria (18.0-28.0%). Reads mapping showed that exDNA read depths were similar across all predicted open reading frames from assembled genomes that were assigned as Bacteroidetes which is consistent with cell lysis as a source of exDNA. Enrichment of CRISPR-CAS proteins in exDNA reads and CRISPR spacers in Bacteroidetes associated draft genomes suggested that bacteriophage infection may be an important cause of lysis of these cells. A critical role for this exDNA was found using DNase I digestion experiments which showed that the exDNA was vital for the structural stability of relatively small sized AGS but not for the larger sized AGS. The characteristics of exDNA in AGSs revealed in this work provide a new perspective on AGS components and structural stability.

Genome-based analysis to understanding rapid resuscitation of cryopreserved anammox consortia via sequential supernatant addition.

Simple cryopreservation of anaerobic ammonium-oxidation (anammox) consortia has become a promising preservation technology for the fast start-up of the anammox process. Here, we use genome-resolved metagenomics and metatranscriptomics to understand of the microbial interaction in a simple and effective resuscitation process for long-term cryopreserved anammox consortia by sequential addition of anammox SBR supernatant. Performance results showed that sequential addition of anammox supernatant significantly reduced the resuscitation time of the granule-based anammox process from 40 to 20 days. Genome-centric metagenomics were used to recover 19 high-quality draft genomes of anammox and heterotrophic bacteria. Comparative metatranscriptomic analysis was conducted to examine the gene expression of Candidatus Kuenenia stuttgartiensis, the dominant anammox bacterium, and heterotrophic bacteria to better understand their potential interactions. Proteobacteria-affiliated bacteria found in the supernatant were highly active in producing the secondary metabolites molybdopterin cofactor and folate which are needed for growth of the auxotrophic anammox bacteria. In addition, the significantly higher expression levels of hzsA and CO2-fixtion genes in the Candidatus Kuenenia genome indicated the anammox bacteria were likely more active and growing faster after sequential anammox supernatant addition during the resuscitation process. The resuscitation treatment pulse assays confirmed that sequential addition of supernatant was an effective way for the rapid resuscitation of anammox consortia. Our findings offer the first evidence of cross-feeding during the rapid resuscitation of cryopreserved anammox consortia.