Optimizing start-up strategies for the two-inflow nitritation/anammox process: Influence on biofilm microbial community composition.

Low-energy nitrogen removal from ammonium-rich wastewater is crucial in preserving the water environment. A one-stage nitritation/anammox process with two inflows treating ammonium-containing wastewater, supplied from inside and outside the wound filter, is expected to stably remove nitrogen. Laboratory-scale reactors were operated using different start-up strategies; the first involved adding nitritation inoculum after anammox biomass formation in the filter, which presented a relatively low nitrogen removal rate (0.171 kg N/m3 · d), at a nitrogen loading rate of 1.0 kg N/m3 · d. Conversely, the second involved the gradual cultivation of anammox and nitritation microorganisms, which increased the nitrogen removal rate (0.276 kg N/m3 · d). Furthermore, anammox (Candidatus Brocadia) and nitritation bacteria (Nitrosomonadaceae) coexisted in the biofilm formed on the filter surface. The abundance of nitritation bacteria (10.5%) in the reactor biofilm using the second start-up strategy was higher than that using the first (3.7%). Thus, the two-inflow nitritation/anammox process effectively induced habitat segregation using a suitable start-up strategy.

Microbial diversity in shallow-sea sediment from Tsukumo Bay, Japan, determined by 16S rRNA gene amplicon sequencing.

Information about the microbiota in marine sediments is important because the microbiota and their activities in sediments affect the surrounding marine environment. To evaluate the microbial diversity, we performed 16S rRNA gene amplicon sequencing on sediment samples from 19 stations in Tsukumo Bay, the northern area of Noto Peninsula, Japan.

Antimicrobial resistome and mobilome in the urban river affected by combined sewer overflows and wastewater treatment effluent.

The dissemination of antimicrobial resistance in the environment is an emerging global health problem. Wastewater treatment effluent and combined sewer overflows (CSOs) are major sources of antimicrobial resistance in urban rivers. This study aimed to clarify the effect of municipal wastewater treatment effluent and CSO on antimicrobial resistance genes (ARGs), mobile gene elements, and the microbial community in an urban river. The ARG abundance per 16S-based microbial population in the target river was 0.37-0.54 and 0.030-0.097 during the CSO event and dry weather, respectively. During the CSO event, the antimicrobial resistome in the river shifted toward a higher abundance of ARGs to clinically important drug classes, including macrolide, fluoroquinolone, and ß-lactam, whereas ARGs to sulfonamide and multidrug by efflux pump were relatively abundant in dry weather. The abundance of intI1 and tnpA genes were highly associated with the total ARG abundance, suggesting their potential application as an indicator for estimating resistome contamination. Increase of prophage during the CSO event suggested that impact of CSO has a greater potential for horizontal gene transfer (HGT) via transduction. Consequently, CSO not only increases the abundance of ARGs to clinically important antimicrobials but also possibly enhances potential of HGT in urban rivers.

A quantitative sequencing method using synthetic internal standards including functional and phylogenetic marker genes.

The method of spiking synthetic internal standard genes (ISGs) to samples for amplicon sequencing, generating sequences and converting absolute gene numbers from read counts has been used only for phylogenetic markers and has not been applied to functional markers. In this study, we developed ISGs, including gene sequences of the 16S rRNA, pmoA, encoding a subunit of particulate methane monooxygenase and amoA, encoding a subunit of ammonia monooxygenase. We added ISGs to the samples, amplified the target genes and performed amplicon sequencing. For the mock community, the copy numbers converted from read counts using ISGs were equivalent to those obtained by the quantitative real-time polymerase chain reaction (4.0 × 104 versus 4.1 × 104 and 3.0 × 103 versus 4.0 × 103 copies µL-DNA-1 for 16S rRNA and pmoA genes, respectively), but we also identified underestimation, possibly due to primer coverage (7.8 × 102 versus 3.7 × 103 µL-DNA-1 for amoA gene). We then applied this method to environmental samples and analysed phylogeny, functional diversity and absolute quantities. One Methylocystis population was most abundant in the sludge samples [16S rRNA gene (3.8 × 109 copies g-1 ) and the pmoA gene (2.3 × 109 copies g-1 )] and were potentially interrelated. This study demonstrates that ISG spiking is useful for evaluating sequencing data processing and quantifying functional markers.

Ammonia tolerance and microbial community in thermophilic co-digestion of sewage sludge initiated with lignocellulosic biomass.

Rice straw is a useful lignocellulosic biomass for controlling ammonia inhibition in the thermophilic anaerobic digestion of sewage sludge. However, it is challenging to procure rice straw throughout the year because of its seasonal production. This study investigated methane production in a laboratory-scale digester by gradually decreasing rice straw addition to solid thermophilic sewage sludge digestion. The decrease in rice straw did not accumulate volatile fatty acids and stabilized methane production. Even with increased sludge concentration without rice straw, methane production continued under high ammonia conditions. Ammonia tolerance of the digested sludge of the experimental digester was higher than that of conventionally digested sludge. The cellulose-degrading bacteria Clostridia and high ammonia-resistant archaea Methanosarcina were dominant in the experimentally digested sludge. The community was maintained for over 200 days after discontinuing the rice straw supply. These findings suggest that anaerobic digestion initiation with rice straw is appropriate to facilitate ammonia-tolerant communities.

Environmental Factors Affecting the Community of Methane-oxidizing Bacteria.

Methane-oxidizing bacteria (MOB) are ubiquitous and play an important role in the mitigation of global warming by reducing methane. MOB are commonly classified into Type I and Type II, belonging to Gammaproteobacteria and Alphaproteobacteria, respectively, and the diversity of MOB has been examined. However, limited information is currently available on favorable environments for the respective MOB. To investigate the environmental factors affecting the dominant type in the MOB community, we performed MOB enrichment using down-flow hanging sponge reactors under 38 different environmental conditions with a wide range of methane (0.01-80%) and ammonium concentrations (0.001-2,000| |mg N L-1) and pH 4-7. Enrichment results revealed that pH was a crucial factor influencing the MOB type enriched. Type II was dominantly enriched at low pH (4-5), whereas Type I was dominant around neutral pH (6-7). However, there were some unusual cultivated biomass samples. Even though high methane oxidation activity was observed, very few or zero conventional MOB were detected using common FISH probes and primer sets for the 16S rRNA gene and pmoA gene amplification. Mycobacterium mostly dominated the microbial community in the biomass cultivated at very high NH4+ concentrations, strongly implying that it exhibits methane oxidation activity. Collectively, the present results revealed the presence of many unknown phylogenetic groups with the capacity for methane oxidation other than the reported MOB.

Effects of organic carbon and sulfide on the anammox reaction in the anoxic column in the SRDAPN process for treating high-strength wastewater.

Low energy consumption treatment of high-strength wastewater is crucial in controlling groundwater pollution and eutrophication in closed waterbodies. In this study, the sulfate reduction, denitrification/anammox, and partial nitrification (SRDAPN) process, which is an effective organic carbon and nitrogen removal process with low energy consumption for low strength wastewater, was applied to treat livestock wastewater with high COD and sulfate concentration, and microbial reaction and community were examined using an anaerobic-anoxic biological filter reactor that simulates circulation from an aerobic reactor. At a total organic carbon loading rate of 2.7-5.8 kgC/m3·day, sulfate reduction and methane production occurred simultaneously in the anaerobic column of the reactor. Specifically, sulfate reduction resulted in organic matter removal rates of 38 and 26% at ambient temperature and 25 °C, respectively. Furthermore, both heterotrophic and autotrophic denitrification occurred in the anoxic column, and when the organic loading rate in the anoxic reactor was below 0.2 kgC/m3·day, 33%-37% of ammonium and 33%-34% of nitrite were removed by the anammox reaction. Heterotrophic denitrification bacteria (Thauera, Comamonas, and Denitratisoma) and sulfur denitrification bacteria (Sulfurimonas denitrificans) grew in the lower and middle parts of the anoxic column, whereas anammox bacteria (2.5% of Candidatus Brocadia at ambient temperature and 9.4% of Candidatus Kuenenia at 25 °C) grew in the upper part of the anoxic column. These results indicate that the SRDAPN process based on sulfur cycle and anammox is useful for treatment of high strength wastewater with low energy consumption.

Distinct comammox Nitrospira catalyze ammonia oxidation in a full-scale groundwater treatment bioreactor under copper limited conditions.

Microbial ammonia oxidation is the initial nitrification step used in biological nitrogen-removal during water treatment processes, and the discovery of complete ammonia-oxidizing (comammox) bacteria added a novel member to this functional group. It is important to identify and understand the predominant microorganisms responsible for ammonium removal in biotechnological process design and optimization. In this study, we used a full-scale bioreactor to treat ammonium in groundwater (9.3 ± 0.5 mg NH4+-N/L) and investigated the key ammonia-oxidizing prokaryotes present. The groundwater ammonium was stably and efficiently oxidized throughout ∼700 days of bioreactor operation. 16S rRNA gene amplicon sequencing of the bioreactor community showed a high abundance of Nitrospira (12.5-45.9%), with the dominant sequence variant (3.5-37.8%) most closely related to Candidatus Nitrospira nitrosa. Furthermore, analyses of amoA, the marker gene for ammonia oxidation, indicated the presence of two distinct comammox Nitrospira populations, however, the relative abundance of only one of these populations was strongly correlated to ammonia oxidation rates and was robustly expressed. After 380 days of operation copper wires were immersed into the reactor at 0.04-0.06 m2/m3 tank, which caused a gradual abundance increase of one discrete comammox Nitrospira population. However, further increase of the copper dosing (0.08 m2/m3 tank) inverted the most abundant ammonia-oxidizing population to Nitrosomonas sp. These results indicate that comammox Nitrospira were capable of efficient ammonium removal in groundwater without exogenous nutrients, but copper addition can stimulate comammox Nitrospira or lead to dominance of Nitrosomonas depending on dosage.

Sediment Microbiota in Response to Circuitry of Sediment Microbial Fuel Cells, Revealed by 16S rRNA Gene Amplicon Sequencing.

Information about sediment microbiota affected by sediment microbial fuel cells (SMFC) is limited. A laboratory-scale SMFC was applied to a eutrophic lake sediment under closed-circuit/open-circuit conditions. We analyzed the prokaryotes in the sediment adhering to the anode material. The archaeal family Methanoperedenaceae was a predominant group under closed-circuit conditions.

Detection of potentially pathogenic Arcobacter spp. in Bangkok canals and the Chao Phraya River.

The management of pathogenic bacteria in waterways is a public health issue. Here, we investigated the concentrations of potentially pathogenic bacteria, Arcobacter spp. and Campylobacter spp., and Escherichia coli, by quantifying species-specific genes in surface water samples from canals and the Chao Phraya River from June 2017 to June 2018 in Bangkok, Thailand. We assessed the relationship between the specific bacterial concentrations, water quality, and seasonal changes. Arcobacter spp. were detected at high density in all samples and showed seasonal fluctuations according to analyses based on 16S rDNA and the invasion gene ciaB. High levels of 16S rDNA and dut gene of E. coli were detected in the polluted drainage canals. A high correlation was observed between E. coli and chemical and biochemical oxygen demand (COD and BOD), suggesting that untreated domestic wastewater was the source of the E. coli. In contrast, Arcobacter spp. were detected with high density even in water samples with relatively low COD, suggesting that Arcobacter spp. are more likely than E. coli to survive in the water environment. The analysis of 16S rDNA and ciaB gene sequence analyses indicated that the Arcobacter spp. isolated from the drainage canals were A. butzleri and A. cryaerophilus.

Initial behaviors and removal of extracellular plasmid gene in membrane bioreactor.

Extracellular antibiotic resistance genes (eARG) are considered to play an important role in spread of antimicrobial resistance (AMR) in wastewater treatment and water environment. Membrane bioreactor (MBR) reportedly has better removal of ARGs in wastewater than conventional activated sludge process. However, removal of eARG is possibly limited because eARG is small to pass through microfiltration (MF) membranes. To evaluate potential removal of eARG in MBR, this study aimed to understand the initial behaviors of eARG received in MBR. The recombinant plasmid with artificial marker gene was spiked in lab-scale MBR to trace fate of eARG in MBR. Among 10 10 copies/L of the spiked gene, 2.6 × 109 copies/L was adsorbed on sludge particles at 6 h after spiking, while only 2.2 × 108-3.6 × 108 copies/L of the spiked gene was remained but constant in sludge liquid phase from 6 until 48 h. This result suggests that adsorption on sludge particles served as the main mechanism to govern the initial fate of eARG in MBR. Meanwhile, the spiked gene concentrations in membrane permeate was lower than sludge liquid phase and decreased overtime, suggesting retention of eARG in membrane filtration. Total LRV of the spiked extracellular gene were 3.4 ± 0.8 log at 48 h after spiking. LRV by adsorption corresponded to 1.7 ± 0.7 log constantly since 3 h after spiking, while LRV by membrane filtration increased from 0 to 1.7 ± 0.6 log. Linear correlation of LRV by membrane filtration with transmembrane pressure (TMP) suggested that foulant deposition on membrane governs removal of eARG by membrane filtration in MBR.

Promotion of biological nitrogen fixation activity of an anaerobic consortium using humin as an extracellular electron mediator.

Nitrogen fertiliser is manufactured using the industrial Haber-Bosch process, although it is extremely energy-consuming. One sustainable alternative technology is the electrochemical promotion of biological nitrogen fixation (BNF). This study reports the promotion of BNF activity of anaerobic microbial consortia by humin, a solid-phase humic substance, at any pH, functioning as an extracellular electron mediator, to levels of 5.7-11.8 times under nitrogen-deficient conditions. This was evidenced by increased acetylene reduction activity and total nitrogen content of the consortia. Various humins from different origins promoted anaerobic BNF activity, although the degree of promotion differed. The promotion effected by humin differed from the effects of chemical reducing agents and the effects of supplemental micronutrients and vitamins. The promotion of anaerobic BNF activity by only reduced humin without any other electron donor suggested that humin did not serve as organic carbon source but as extracellular electron mediator, for electron donation to the nitrogen-fixing microorganisms. The next generation sequencing (NGS) of partial 16S rRNA genes showed the predominance of Clostridiales (Firmicutes) in the consortia. These findings suggest the effectiveness of humin as a solid-phase extracellular electron mediator for the promotion of anaerobic BNF activity, potentially to serve for the basis for a sustainable technology.

Metagenomic insights into the effect of sulfate on enhanced biological phosphorus removal.

Excess phosphorus in water supplies causes eutrophication, which degrades water quality. Hence, the efficient removal of phosphorus from wastewater represents a highly desirable process. Here, we evaluated the effect of sulfate concentration on enhanced biological phosphorus removal (EBPR), in which phosphorus is typically removed under anaerobic-oxic cycles, with sulfate reduction the predominant process in the anaerobic phase. Two sequencing batch EBPR reactors operated under high- (SBR-H) vs. low-sulfate (SBR-L) concentrations for 189 days and under three periods, i.e., start-up, sufficient acetate, and limited acetate. Under acetate-rich conditions, phosphorus removal efficiency was > 90% for both reactors; however, under acetate-limited conditions, only 34% and 91.3% of the phosphorus were removed for the SBR-L and the SBR-H, respectively. Metagenomic sequencing of the reactors showed that the relative abundance of the polyphosphate-accumulating and sulfur-reducing bacteria (SRB) was higher in the SBR-H, consistent with its higher phosphorus removal activity. Ten high-quality metagenome-assembled genomes, including one closely related to the genus Thiothrix disciformis (99.81% average amino acid identity), were recovered and predicted to simultaneously metabolize phosphorus and sulfur by the presence of phosphorus (ppk, ppx, pst, and pit) and sulfur (sul, sox, dsr, sqr, apr, cys, and sat) metabolism marker genes. The omics-based analysis provided a holistic view of the microbial ecosystem in the EBPR process and revealed that SRB and Thiothrix play key roles in the presence of high sulfate.Key points• We observed high phosphorus-removal efficiency in high-sulfate EBPR.• Metagenome-based analysis revealed sulfate-related metabolic mechanisms in EBPR.• SRB and PAOs showed interrelationships in the EBPR-sulfur systems.

A bioelectrochemical-system-based trickling filter reactor for wastewater treatment.

A bioelectrochemical system (BES)-based trickling filter (TF) reactor was utilized for wastewater treatment. At a COD load of 1.0 g-COD/L/day, effluent chemical oxygen demand (COD) and total nitrogen (TN) were 115 and 108 mg/L, respectively, which were allowed for discharge. Superior performance was achieved at 0.5 g-COD/L/day with a circulation rate of 8 L/h, and both COD and TN removal were >98%. Coulombic efficiency was 11% at 1.0 g-COD/L/day and at most 16% at 0.5 g-COD/L/day. COD removal decreased when the BES was removed, demonstrating that BES improved COD removal capability. In anodic biofilms, exoelectrogenic, facultative, nitrifying, and sulfate-reducing bacteria could coexist. Geobacter for current generation grew inside the biofilm, and bacteria in the middle and outer layers consumed oxygen and degraded organic matter and nitrogen. This BES-based TF reactor may be used for efficient and cost-effective COD and TN removal at high loads without excess sludge removal.

Wastewater Treatment using the "Sulfate Reduction, DenitrificationAnammox and Partial Nitrification (SRDAPN)" Process.

Although nitrogen removal from wastewater is essential to prevent eutrophication, the biological processes employed to this end are characterized by several disadvantages, including high energy consumption and the production of large quantities of sludge. Thus, in this study, the organic matter and nitrogen removal efficiencies of the new sulfate reduction, denitrification/anammox and partial nitrification (SRDAPN) process were examined using an anaerobic-anoxic-oxic biofilter reactor. The results showed that the organic matter removal efficiency of the new process at loading rate 1.0 kg COD/m3 per day was 97%. With a circulation flow from the oxic to the anoxic column that was 3 times influent, the nitrogen removal efficiency of the sulfur denitrification and nitrification (SRDN) process without anammox, was 66%, while that of the SRDAPN process with anammox was 76%. Additionally, nitrogen consumption by the anammox reaction in the anoxic column was 13.8% for nitrite-nitrogen and 10.5% for ammonium-nitrogen, and the withdrawal of excess sludge was not required throughout the 170 days of operation. Microbial community analysis showed that acetogenic sulfate reducing bacteria and acetoclastic methanogens coexisted in the anaerobic column, and in the anoxic column, the total relative abundance of anammox bacteria, including Candidatus Brocadia, which coexisted with heterotrophic denitrifying bacteria and sulfur denitrifying bacteria, was 17-18%. Thus, this study established the SRDAPN process as an energy saving and high removal efficiency process.

Biodegradability of woody film produced by solvent volatilisation of Japanese Beech solution.

To address the problem of marine pollution from discarded plastics, we developed a highly biodegradable woody film, with almost the same components as wood, from the formic acid solution of ball-milled wood. We found that the woody film was not easily degraded by cultured solution of hand bacteria (phylum Proteobacteria was dominant). However, the film was easily biodegraded when in cultured solution of soil (Firmicutes, especially class Bacilli, was dominant) for 4 weeks at 37 °C, or when buried in the soil itself, both under aerobic conditions (Acidobacteria and Proteobacteria were dominant) for 40 days at room temperature and under anaerobic conditions (Firmicutes, especially family Ruminococcaceae, was dominant) for 5 weeks at 37 °C. Moreover, when film was buried in the soil, more carbon dioxide was generated than from soil alone. Therefore, the film was not only brittle but formed of decomposable organic matter. We showed that the film does not decompose at the time of use when touched by the hand, but it decomposes easily when buried in the soil after use. We suggest that this biodegradable woody film can be used as a sustainable raw material in the future.

Enhancement of methane production and phosphorus recovery with a novel pre-treatment of excess sludge using waste plaster board.

A new pre-treatment process for excess sludge is proposed to increase methane production and recover phosphorus by adding waste plaster board as calcium sulfate. The content of calcium sulfate in the plaster granules (PG) used in this study is 99%. When PG and calcium sulfate are added to the excess sludge generated from a municipal wastewater treatment plant, acetate production is enhanced as per sulfate reduction and phosphorus release is reduced via the formation of calcium phosphate. In the continuous pre-treatment experiment performed at 25 °C and for 10 days of sludge retention time (SRT) using calcium sulfate, 1935 ±â€¯395 mg/L of acetate is produced with 1070 ±â€¯255 mg/L of sulfate, which is reduced. Desulfobulbus spp., which can oxidize organic matter to acetate incompletely, have been observed in the pre-treated sludge. The pre-treated sludge has subsequently been used for methophiric anaerobic digestion. The methane yield from the pre-treated sludge is found to be 1.2 times that of the non-pretreated sludge at an SRT of 30 days, indicating that the pre-treatment using PG can improve methane production. Phosphorus is released from the non-pretreated sludge in the digester. Nevertheless, a decrease in phosphorus content has been observed, resulting in the digested sludge containing calcium phosphate that is useful for agriculture.

Candidates of quorum sensing bacteria in activated sludge associated with N-acyl homoserine lactones.

This study aimed to explore candidates of microbial groups which is associated with quorum sensing in activated sludge. Activated sludge samples were collected from three wastewater treatment plants (WWTP) to analyze N-acyl homoserine lactone (AHL) by Fourier-transform mass spectrometry (FTMS) and 16S rRNA-based microbial community. Among activated sludge samples taken at 3 WWTPs in different seasons, 2 AHL species of N-3-hydroxyoctanoyl-l-homoserine lactone and N-3-hydroxydecanoyl-l-homoserine lactone were detected in the range of ranged of 0.1 ng/L to 1.6 ng/L. The detected AHL species were not dependent on treatment systems nor seasons. From microbial community analysis, population abundance of one strain in Verrucomicrobia and two strains in Holophagaceae had high correlation with AHL concentration in activated sludge. Comamonadaceae had also moderately correlated population with AHL concentrations among quorum sensing bacteria reported previously.

Influence of tetramethylammonium hydroxide (TMAH) on the microbial properties of anaerobic granular sludge acclimated to isoplophyl alcohol (IPA) wastewater under psychrophilic conditions.

In this study, a continuous flow experiment was conducted in which a lab-scale upflow anaerobic sludge blanket (UASB) reactor at psychrophilic conditions (18-19°C) was fed with artificial wastewater, containing tetramethylammonium hydroxide (TMAH) and isoplophyl alcohol (IPA), from the electronics industry. This was done to evaluate process performance and microbial properties of the granular sludge that was retained in the reactor. The inoculated granular sludge was precultured with IPA containing wastewater but not TMAH; as a result, no degradation was observed in 30 days of operation. To enhance degradation, the reactor was seeded with 2% weight of the TMAH-enriched sludge, after which TMAH was enhanced. Consequently, the total COD removal efficiency reached 90% at an organic loading rate of 7.5 kg COD/m3/day. The TMAH inflow decreased the diameter of the retained granular sludge, but the sludge retained its settleability. The proliferation of the Methanometylovorans microorganisms present in the enrichment culture was confirmed by analysis of the 16 S rRNA gene in the retained sludge. In addition, TMAH degradation was inhibited by addition chloroform, a methanogen inhibitor. These results suggested species in the genus Methanometylovorans in the granular sludge contributed significantly to methanogenic TMAH degradation.

Optimization of rotational speed and hydraulic retention time of a rotational sponge reactor for sewage treatment.

A rotational sponge (RS) reactor was proposed as an alternative sewage treatment process. Prior to the application of an RS reactor for sewage treatment, this study evaluated reactor performance with regard to organic removal, nitrification, and nitrogen removal and sought to optimize the rotational speed and hydraulic retention time (HRT) of the system. RS reactor obtained highest COD removal, nitrification, and nitrogen removal efficiencies of 91%, 97%, and 65%, respectively. For the optimization, response surface methodology (RSM) was employed and optimum conditions of rotational speed and HRT were 18 rounds per hour and 4.8 h, respectively. COD removal, nitrification, and nitrogen removal efficiencies at the optimum conditions were 85%, 85%, and 65%, respectively. Corresponding removal rates at optimum conditions were 1.6 kg-COD m-3d-1, 0.3 kg-NH4+-N m-3d-1, and 0.12 kg-N m-3d-1. Microbial community analysis revealed an abundance of nitrifying and denitrifying bacteria in the reactor, which contributed to nitrification and nitrogen removal.