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.

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.

Spatiotemporal distribution of veterinary and human drugs and its predictability in Japanese catchments.

Little is known about the predictability of mass flows of veterinary drugs in Asian catchments, where effluent from livestock farms is a major source. We therefore conducted this study to understand the applicability and limitations of a population-based emission model, which assumed usage of veterinary and human drugs to be evenly distributed over the national livestock or human population throughout the year, and sources to be effluent discharges at livestock farms, households, and sewage treatment plants in Japanese catchments. We monitored five veterinary drugs (lincomycin, sulfamonomethoxine, tiamulin, tylosin, and tilmicosin), two human and livestock drugs (sulfamethoxazole and trimethoprim), two human drugs (carbamazepine and clarithromycin), and a metabolite (sulfapyridine) of a human drug once a month over 2 years in eight Japanese rivers which have active livestock farming in their catchments. Mass flows of carbamazepine and sulfapyridine were stable, while those of veterinary drugs fluctuated widely, especially sulfamonomethoxine and tilmicosin, whose 25 %-100 % ranges averaged 1.5 and 1.2 log units, respectively, attributable mainly to their usage patterns. The model accurately predicted mean mass flows of carbamazepine in the rivers with errors of <±0.3 log unit. Although it slightly to moderately overestimated those of the other four human-related compounds, the incorporation of an empirical correction factor, determined to minimize mean absolute error (MAE) among the rivers, substantially lowered their MAEs to <0.23 log units. However, the MAEs of the five veterinary drugs were as high as 0.42 (sulfamonomethoxine) to 0.60 (tiamulin) log units even with the coefficient, likely due mainly to the spatial distribution of their usage per capita. So as not to overlook spatiotemporal elevation of risks of veterinary drugs, a stochastic method should be applied in their management. This is the first study to assess the use of spatiotemporal homogeneity in usage per capita of veterinary drugs in Asian catchments.

In-stream sorption of azithromycin and levofloxacin in a river receiving sewage treatment plant effluent.

Modelling natural attenuation is crucial to managing pharmaceuticals. However, little is known about the mechanism behind their in-stream sorption. To better understand the in-stream attenuation of the highly sorptive antibiotics azithromycin (AZM) and levofloxacin (LVF), we monitored them in a 2.1-km stretch of the Asano River under diverse flow conditions. This stretch receives effluent directly from a sewage treatment plant (STP), which was a dominant source of the pharmaceuticals. Average distribution coefficients between dissolved and particulate phases (Kd,SPM) in the outflow river water were 6.3×105 L/kg for AZM and 7.5×104 L/kg for LVF, while those in the STP effluent were 1-2 orders of magnitude lower. Mass balances in the river stretch calculated by considering only dissolved phase (MBw) and both dissolved and particulate phases (MBs) were 8%-52% and 58%-102%, respectively, for AZM, and 58%-71% and 60%-105% for LVF. MBw

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.

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.

Fate and seasonal change of Escherichia coli resistant to different antibiotic classes at each stage of conventional activated sludge process.

This study investigated the impact of each treatment stage of the activated sludge process on the fate of antibiotic resistant bacteria (ARB) in wastewater treatment plants (WWTPs). Wastewater and sludge samples were collected monthly at each stage of a commercial-scale WWTP. After 20-25 strains of indicator Escherichia coli were isolated from each sample on Chromocult Coliform Agar, antibiotic resistance of the isolates to amoxicillin (AMX), ciprofloxacin (CIP), norfloxacin (NFX), kanamycin (KM), sulfamethoxazole/trimethoprim (ST) and tetracycline (TC) were tested with the Kirby-Bauer disk diffusion method. As a result, activated sludge in the aeration tank and return sludge had higher abundance of antibiotic resistant E. coli than influent wastewater and secondary treatment effluent. AMX resistant E. coli was enriched in return sludge at the secondary clarifier. Higher temperature was also likely to cause an increase of AMX resistant E. coli in sludge. The antibiotic resistance profile of E. coli in secondary treatment effluent was more dependent on activated sludge than influent wastewater. These results suggested that activated sludge in WWTP possibly serves as a reservoir of ARB, and that behavior of ARB in WWTP differs by antibiotic classes.

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.

Flame-Oxidized Stainless-Steel Anode as a Probe in Bioelectrochemical System-Based Biosensors to Monitor the Biochemical Oxygen Demand of Wastewater.

Biochemical oxygen demand (BOD) is a widely used index of water quality in wastewater treatment; however, conventional measurement methods are time-consuming. In this study, we analyzed a novel flame-oxidized stainless steel anode (FO-SSA) for use as the probe of bioelectrochemical system (BES)-based biosensors to monitor the BOD of treated swine wastewater. A thinner biofilm formed on the FO-SSA compared with that on a common carbon-cloth anode (CCA). The FO-SSA was superior to the CCA in terms of rapid sensing; the response time of the FO-SSA to obtain the value of R2 > 0.8 was 1 h, whereas the CCA required 4 h. These results indicate that the FO-SSA offers better performance than traditional CCAs in BES biosensors and can be used to improve biomonitoring of wastewater.

Improvement of dewatering characteristics by co-digestion of rice straw with sewage sludge.

A continuous mesophilic co-digestion of sewage sludge and softened rice straw was conducted and the dewatering characteristics of digested sludge were evaluated by a dewatering experiment using a belt press. The digestion was operated with solid retention time (SRT) of 25 days, and the feeding ratio of sludge to rice straw was 1:0.5 (total solids base). After 129 days of stable operation, the properties of digested sludge were analysed; then five kinds of cationic coagulants were tested to select the optimal coagulants for dewatering, and two coagulants were selected and used in the dewatering experiment because of lower doses and lower moisture of sludge cakes. Sludge property analysis showed that by the addition of rice straw, the fibrous materials in the digested sludge increased remarkably and the normalized capillary suction time (CST) decreased significantly, indicating that the dewatering properties was improved. The results of dewatering experiment showed that by the addition of rice straw, specific filtration rate of digested sludge increased by 81.2% and 174.6%, respectively; water content of dewatered sludge cakes decreased by 8.2% and 13.4%, respectively. The dewaterability of digested sludge was suggested to be improved due to rice straw addition.

Mitigation of nitrous oxide (N2 O) emission from swine wastewater treatment in an aerobic bioreactor packed with carbon fibers.

Mitigation of nitrous oxide (N2 O) emission from swine wastewater treatment was demonstrated in an aerobic bioreactor packed with carbon fibers (CF reactor). The CF reactor had a demonstrated advantage in mitigating N2 O emission and avoiding NOx (NO3 + NO2 ) accumulation. The N2 O emission factor was 0.0003 g N2 O-N/gTN-load in the CF bioreactor compared to 0.03 gN2 O-N/gTN-load in an activated sludge reactor (AS reactor). N2 O and CH4 emissions from the CF reactor were 42 g-CO2 eq/m(3) /day, while those from the AS reactor were 725 g-CO2 eq/m(3) /day. The dissolved inorganic nitrogen (DIN) in the CF reactor removed an average of 156 mg/L of the NH4 -N, and accumulated an average of 14 mg/L of the NO3 -N. In contrast, the DIN in the AS reactor removed an average 144 mg/L of the NH4 -N and accumulated an average 183 mg/L of the NO3 -N. NO2 -N was almost undetectable in both reactors.

Nitrogen and phosphorus removal from wastewater treatment plant effluent via bacterial sulfate reduction in an anoxic bioreactor packed with wood and iron.

We investigated the removal of nitrogen and phosphate from the effluent of a sewage treatment plant over a long-term operation in bioreactors packed with different combinations of wood and iron, with a trickling filter packed with foam ceramics for nitrification. The average nitrification rate in the trickling filter was 0.17 kg N/m3∙day and remained at 0.11 kg N/m3∙day even when the water temperature was below 15 °C. The denitrification and phosphate removal rates in the bioreactor packed with aspen wood and iron were higher than those in the bioreactor packed with cedar chips and iron. The bioreactor packed with aspen wood and iron continued to remove nitrate and phosphate for >1200 days of operation. The nitrate removal activity of a biofilm attached to the aspen wood from the bioreactor after 784 days of operation was 0.42 g NO3-N/kg dry weight wood∙ day. There was no increase in the amount of dissolved organic matter in the outflow from the bioreactors.

Sulfate-reducing bacteria in a denitrification reactor packed with wood as a carbon source.

A denitrification reactor packed with wood as a carbon source was operated using synthetic inorganic wastewater. The maximum denitrification rate was 62.4 g-NO(3)(-)/m(3)/day at HRT of 24 h. The nitrate removal continued after 1500 days. The denitrification efficiency was assumed to enhance sulfur denitrification via wood degradation by sulfate reduction. The achieved sulfate reduction rate was 468 mg-SO(4)(2-)/kg-dry weight wood/day after 419 days of operation. The sulfate reduction rate in the deep-layer biofilm inside the wood was higher than that in the total biofilm inside the wood. The sulfate-reducing bacteria segregated inside the wood. This study suggested that Desulfobulbus spp. and Desulfomicrobium spp. grown in the deep-layer degraded the wood incompletely, and the produced organic acids were utilized by the heterotrophic denitrifying bacteria, Desulfobacter spp. and Desulfonema spp., grown in the surface layer, and that these surface bacteria complete the degradation of the organic acids from the wood.

Phosphate removal and sulfate reduction in a denitrification reactor packed with iron and wood as electron donors.

Phosphorus removal and denitrification using iron and wood as electron donors were examined in a laboratory-scale biological filter reactor. Phosphorus removal and denitrification using iron and wood continued for 1,200 days of operation. Wood degradation by heterotrophic denitrification and iron oxidation by hydrogenotrophic denitrification occurred simultaneously. In the biofilm inside the wood, not only heterotrophic denitrification activity but also sulfate reduction and sulfur denitrification activities were recognized inside the wood, indicating that a sulfur oxidation-reduction cycle was established. Sulfate reduction and denitrification were accelerated with the addition of cellulose. Microbial communities of sulfate-reducing bacteria by PCR primer sets could be amplified in the biofilm in the reactors. The dissimilatory sulfite reductase gene and the 16S rRNA gene of six phylogenetic groups of SRB in the reactors were analyzed. Some SRB group-specific primers-amplification products were obtained inside the wood and around iron.