Adaptation of anammox granules in swine wastewater treatment to low temperatures at a full-scale simultaneous partial nitrification, anammox, and denitrification plant.

In the anammox process, maintaining a high anammox activity at low water temperatures for stable nitrogen removal is a challenge. In this study, to verify the adaptability of anammox to low water temperatures, we investigated effects of annual temperature fluctuations on nitrogen removal in a full-scale swine wastewater treatment plant, where anammox bacteria accumulated. Annual quarters were defined as L-1 (November-January), L-2 (February-April), H-1 (April-July), and H-2 (July-October). The total nitrogen removal rate was stable at 0.08-0.11 kg-N/m3/d, even during temperature fluctuations. Removal efficiencies of biochemical oxygen demand and total nitrogen were consistently high at 95-99% and 69-81%, respectively. The anammox activity and abundance of anammox bacteria were highest in granule L-1 and lowest in granule H-2. The optimal temperature for anammox activity shifted from 35 °C in granules H-1 and H-2 to 30 °C in granules L-1 and L-2, while the latter maintained a moderate activity compared to the former at low temperature. Candidatus Jettenia asiatica was predominant, especially in granule L-2, accounting for up to 54% of the microbial community composition at the genus level. The high specific anammox activity in granule L-2 was considered to be due to the abundance of anammox bacteria and the adaptation of Ca. Jettenia asiatica to low temperature. The anammox granules adapted well to low temperatures and demonstrated high efficiency in the simultaneous partial nitrification anammox and denitrification process without heating. Thus, constructing an energy-saving and cost-effective nitrogen removal system can be considered.

Full-scale simultaneous partial nitrification, anammox, and denitrification process for treating swine wastewater.

A full-scale swine-wastewater activated sludge treatment plant that contains naturally enriched anammox biofilms was investigated for 2 years. Red biofilm in this system included Planctomycetes at a maximum of 62.5% of the total bacteria diversity, including Candidatus Jettenia and Candidatus Brocadia. The plant was operated with an influent containing 1,104 ± 513 mg/L biochemical oxygen demand (BOD) and 629 ± 198 mg/L total nitrogen (TN) (BOD/N of 1.78 ± 0.58) at a volumetric BOD loading rate of 0.32 ± 0.12 kg/m3/d. Notwithstanding drastically varying influent concentrations, BOD removal efficiency was stable at 95 ± 4%. However, TN removal fluctuated at 75 ± 14%. Dissolved oxygen (DO) concentrations in the aeration tank were 0.06-2.0 mg/L. DO concentration greatly affected nitrogen removal, e.g. when DO was lower than 0.3 mg/L, total inorganic nitrogen removal was 61 ± 14% (≤20 °C), 78 ± 16% (20-30 °C), and 75 ± 12% (≥30 °C), whereas at higher DO concentrations, removal rates were 47 ± 13%, 55 ± 16%, and 68%, respectively. As BOD concentration in the influent was limited compared to nitrogen concentration, nitrogen was likely removed by simultaneous nitrification, anammox, and denitrification (SNAD) under microaerobic conditions. Maintaining low DO concentrations would therefore be a simple method to improve nitrogen removal during SNAD processes for swine-wastewater treatment with fluctuating influent.

Effects of thiosulfate addition on ammonia and nitrogen removal in biofilters packed with Oyaishi (pumice tuff).

Ammonia removal is achieved partly by absorption and nitrification in biofilters, resulting in the accumulation of nitrogen and the necessity of treating the effluent water. We investigated the effects of thiosulfate addition to a biofilter containing pumice tuff for ammonia and nitrogen removal in a laboratory-scale experiment. The addition of thiosulfate to the circulating water led to a decreased nitrate and nitrite along with an increase of sulfate. The inorganic nitrogen in the circulating water decreased by up to 44% with thiosulfate addition compared to without thiosulfate. Batch experiments revealed that denitrification activity decreased exponentially along with increases in dissolved oxygen; however, approximately 30% of denitrification activity was maintained at dissolved oxygen concentration of 3.3 mg/L. Metabarcoding of 16S rRNA genes indicated that the genus Thiobacillus had a relative abundance of 0.002%-0.016% of total bacteria in the biofilter packing material. The circulating water pH was decreased below 5 with sulfur oxidation, and ammonium was accumulated without pH control resulting in a decrease in the relative abundance of the family Nitrosomonadaceae. Its relative abundance increased with control of pH to near neutral, indicating that ammonia-oxidizing activity could be maintained by adjusting pH. Thiosulfate addition could stimulate nitrogen removal by sulfur-dependent denitrification in biofiltration systems.

Tolerance of anammox reactor packed with zeolite to partial supply of nitrite or ammonium using purified livestock wastewater.

Anammox reaction requires nitrite and ammonium in the ratio of 1.1-1.3. However, controlling a partial nitrification process prior to the anammox process to maintain this ratio in an influent to the anammox reactor is not easy. In this study, the effect of zeolite on anammox reaction was investigated to determine a method of ammonium preservation in case of partial supply of nitrite or ammonium. Up-flow column type anammox reactors, filled with either zeolite or non-woven fabric, were operated in two-week intervals with purified livestock wastewater containing either ammonium or nitrite. The zeolite reactor showed significantly higher nitrogen removal rates than the non-woven fabric reactor for both influents. When the influent contained ammonium, it was adsorbed onto zeolite, while anammox tolerated starvation for two weeks. In a subsequent reaction cycle, when the influent contained nitrite, anammox used the nitrite and the ammonium desorbed from zeolite. The highest nitrogen removal rates were 0.71 and 0.29 gN/L/day, observed in the zeolite reactor, with the ammonium and nitrite influents, respectively. The limiting factor for reactor performance was zeolite saturation level when the influent contained ammonium and anammox reaction rate when the influent contained nitrite. This study demonstrated that zeolite can buffer the unbalance of the nitrite to ammonium ratio in an anammox reaction, and showed the scopes for improvement under each influent.

Treatment of swine wastewater in continuous activated sludge systems under different dissolved oxygen conditions: Reactor operation and evaluation using modelling.

Swine wastewater was treated in two continuously aerated activated sludge (AS) systems at high (AS1: 1.7-2.6 mg/L) and low (AS2: 0.04-0.08 mg/L) dissolved oxygen (DO), and at three temperatures (10, 20, and 30 °C). Biochemical oxygen demand (BOD) removal was >94.8%. Meanwhile, total nitrogen (N) removal was significantly higher in AS2, at 64, 89, and 88%, than in AS1, at 12, 24, and 46%, for 10, 20, and 30 °C, respectively. The experimental data were considered in a simulation study using an AS model for BOD and N removal, which also included nitrite, free ammonia, free nitrous acid, and temperature. Simulations at high-DO showed that ammonium was partly oxidized into nitrate but not removed, whereas at low-DO ammonium was removed mainly through the nitrite shortcut in simultaneous nitrification-denitrification. This study demonstrates that treatment at low-DO is an effective method for removing N, and modelling a helpful tool for its optimization.

Characterization of the denitrifying bacterial community in a full-scale rockwool biofilter for compost waste-gas treatment.

The potential denitrification activity and the composition of the denitrifying bacterial community in a full-scale rockwool biofilter used for treating livestock manure composting emissions were analyzed. Packing material sampled from the rockwool biofilter was anoxically batch-incubated with 15N-labeled nitrate in the presence of different electron donors (compost extract, ammonium, hydrogen sulfide, propionate, and acetate), and responses were compared with those of activated sludge from a livestock wastewater treatment facility. Overnight batch-incubation showed that potential denitrification activity for the rockwool samples was higher with added compost extract than with other potential electron donors. The number of 16S rRNA and nosZ genes in the rockwool samples were in the range of 1.64-3.27 × 109 and 0.28-2.27 × 108 copies/g dry, respectively. Denaturing gradient gel electrophoresis analysis targeting nirK, nirS, and nosZ genes indicated that the distribution of nir genes was spread in a vertical direction and the distribution of nosZ genes was spread horizontally within the biofilter. The corresponding denitrifying enzymes were mainly related to those from Phyllobacteriaceae, Bradyrhizobiaceae, and Alcaligenaceae bacteria and to environmental clones retrieved from agricultural soil, activated sludge, freshwater environments, and guts of earthworms or other invertebrates. A nosZ gene fragment having 99% nucleotide sequence identity with that of Oligotropha carboxidovorans was also detected. Some nirK fragments were related to NirK from micro-aerobic environments. Thus, denitrification in this full-scale rockwool biofilter might be achieved by a consortium of denitrifying bacteria adapted to the intensely aerated ecosystem and utilizing mainly organic matter supplied by the livestock manure composting waste-gas stream.

Anammox biofilm in activated sludge swine wastewater treatment plants.

We investigated anammox with a focus on biofilm in 10 wastewater treatment plants (WWTPs) that use activated sludge treatment of swine wastewater. In three plants, we found red biofilms in aeration tanks or final sedimentation tanks. The biofilm had higher anammox 16S rRNA gene copy numbers (up to 1.35 × 1012 copies/g-VSS) and higher anammox activity (up to 295 µmoL/g-ignition loss/h) than suspended solids in the same tank. Pyrosequencing analysis revealed that Planctomycetes accounted for up to 17.7% of total reads in the biofilm. Most of them were related to Candidatus Brocadia or Ca. Jettenia. The highest copy number and the highest proportion of Planctomycetes were comparable to those of enriched anammox sludge. Thus, swine WWTPs that use activated sludge treatment can fortuitously acquire anammox biofilm. Thus, concentrated anammox can be detected by focusing on red biofilm.

Utilization of Bacillus sp. strain TAT105 as a biological additive to reduce ammonia emissions during composting of swine feces.

Bacillus sp. strain TAT105 is a thermophilic, ammonium-tolerant bacterium that grows assimilating ammonium nitrogen and reduces ammonia emission during composting of swine feces. To develop a practical use of TAT105, a dried solid culture of TAT105 (5.3 × 10(9) CFU/g of dry matter) was prepared as an additive. It could be stored for one year without significant reduction of TAT105. Laboratory-scale composting of swine feces was conducted by mixing the additive. When the additive, mixed with an equal weight of water one day before use, was added to obtain a TAT105 concentration of above 10(7) CFU/g of dry matter in the initial material, the ammonia concentration emitted was lower and nitrogen loss was approximately 22% lower in the treatment with the additive than in the control treatment without the additive. The colony formation on an agar medium containing high ammonium could be used for enumeration of TAT105 in the composted materials.

Effect of pH on phosphorus, copper, and zinc elution from swine wastewater activated sludge.

With the goal of reducing the amounts of phosphorus (P), copper (Cu), and zinc (Zn) discharged from swine wastewater activated sludge treatment facilities, we studied the elution of these elements from activated sludge at various pH values. Sludge samples with neutral pH collected from three farms were incubated at pH values ranging from 3 to 10. The soluble concentrations of these elements changed dramatically with pH and were highest at pH 3. We assumed that P present in the sludge under neutral and alkaline conditions was in insoluble form bound up with magnesium (Mg) and calcium (Ca), because Ca and Mg also eluted from the sludge at low pH. To clarify forms of Zn and Cu in the sludge, we performed a sequential extraction analysis. Zinc in adsorbed, organically bound, and sulfide fractions made up a large proportion of the total Zn. Copper in organically bound, carbonate, and sulfide fractions made up a large proportion of the total Cu. The soluble P concentrations were lowest at pH 9 or 10 (11-36 mg/L), the soluble Zn concentrations were lowest at pH 8 or 9 (0.07-0.15 mg/L), and the soluble Cu concentrations were lowest at pH 6-9 (0.2 mg/L, the detection limit).

Distribution and characterization of anammox in a swine wastewater activated sludge facility.

Anaerobic ammonium oxidation (anammox) is a novel biological nitrogen removal process that oxidizes NH4(+) to N2 with NO2(-) as an electron acceptor. The purpose of this study was to examine the potential activity and characteristics of anammox in a conventional swine wastewater treatment facility, which uses an activated sludge system consisting of three cascade aeration tanks equipped with ceramic support material. Anammox activity was estimated by a (15)N tracer assay method and was detected in all the sludge and biofilm samples in each aeration tank. Biofilm taken from the third aeration tank, in which the dissolved oxygen concentration was 7.5 mg/L and the wastewater included a high concentration of NO3(-), showed by far the highest anammox activity. A clone library analysis showed the existence of anammox bacteria closely related to 'Candidatus Jettenia asiatica' and 'Ca. Brocadia caroliniensis'. The optimum conditions for anammox activity were a pH of 6.7-7.2, a temperature of 35 °C, a NO2(-) concentration of 10 mmol/L or less, and an NH4(+) concentration of 32 mmol/L or less.

Effect of electron donors on anammox coupling with nitrate reduction for removing nitrogen from nitrate and ammonium.

Anammox coupling with nitrate reduction under various electron donors was studied using sludge acclimatized to have anammox and denitrification activities. Due to a deficiency in electron donors for NO(3)(-) reduction, anammox activity in an inorganic medium containing NO(3)(-) and NH(4)(+) was lower than that in NO(2)(-) and NH(4)(+). Anammox could use NO(2)(-) competitively against denitrifiers under a very limited NO(2)(-) concentration, and additions of swine wastewater or acetate stimulated anammox activity in an inorganic medium containing NO(3)(-) and NH(4)(+) with no inhibition effects. However, a high concentration of swine wastewater caused an exponential increase in denitrification activity. The addition of hydrogen and iron stimulated anammox activity in an inorganic medium containing NO(3)(-) and NH(4)(+), but iron showed an inhibitory effect on anammox in a medium containing NO(2)(-) and NH(4)(+). Hydrogen was shown to be advantageous since it did not increase denitrification even when its addition was increased.

Estimation of Zn and Cu unit output loads from animal husbandry facilities.

Zinc (Zn) and copper (Cu) are toxic to aquatic organisms at very low concentrations that do not affect humans. We measured the daily output of Zn and Cu in wastewater from livestock farms to aquatic environments because waste from animal husbandry operations contains high levels of Zn and Cu. At most pig farms in Japan, a mixture of urine, some faeces, and service water is treated in onsite wastewater treatment facilities and discharged into a water body. Some dairy farms also have wastewater treatment facilities. We surveyed 21 pig farms and six dairy farms. The unit (i.e., per head) output load from piggery wastewater treatment facilities ranged from 0.13 to 17.8 mg/head/d for Zn and from 0.15 to 9.4 mg/head/d for Cu. Over 70% of pig farms had unit output loads of Zn and Cu below 6 and 2 mg/head/d, respectively. For dairy farms, the unit output load from wastewater treatment facilities was estimated at 1.8-3.6 mg/head/d for Zn and 0.6 mg/head/d for Cu. The unit output load for Zn from piggery wastewater treatment facilities was similar to that from treatment facilities for human waste. However, pig farms generally raise several thousand to tens of thousands of pigs; pig farms are therefore presumed to be a significant point source of Zn in rural areas.

Evidence of exponential growth of an anammox population in an anaerobic batch culture.

Twenty-five replicates of growth medium for anaerobic ammonium oxidation (anammox) containing (15)N-labeled ammonium and non-labeled nitrite were inoculated into an anammox enrichment culture at low density, and anaerobically incubated batchwise. In the headspace, (29)N(2) partial pressure linearly increased via anammox in 25 vials, confirming that anammox populations were viable in all subcultures. On prolonged incubation, exponential increases in (29)N(2) were not observed in all but 13 subcultures, suggesting that the anammox population may not proliferate unless all conditions for growth are satisfied. The estimated first-order rate coefficients in those 13 subcultures varied from 0.0029 to 0.0048 h(-1).

Effects of struvite formation and nitratation promotion on nitrogenous emissions such as NH3, N2O and NO during swine manure composting.

To reduce nitrogenous emissions from composting, two different countermeasures were applied simultaneously in swine manure composting. One was forming struvite by adding Mg and P at the start of composting, and the other was to promote nitratation (nitrite being oxidized nitrate) by adding nitrite-oxidizing bacteria after the thermophilic phase of composting. In the laboratory- and mid-scale composting experiments, 25-43% of NH3, 52-80% of N2O and 96-99% of NO emissions were reduced. From the nitrogen balance, it was revealed that the struvite formation reduced not only NH3, but also other nitrogenous emissions except N2O. The amount of total nitrogen losses was reduced by 60% by the two combined countermeasures, against 51% by the struvite formation alone. However, the nitratation promotion dissolved struvite crystals due to the pH decline, diminishing the effect of struvite as a slow-release fertilizer.

Distribution of phosphorus, copper and zinc in activated sludge treatment process of swine wastewater.

Changes in swine wastewater chemical features during an activated sludge treatment process were surveyed on 11 farms, and analyzed with non-biodegradable elements, i.e., phosphorus (P), copper (Cu), and zinc (Zn). In piggery wastewater, they were linearly correlated with suspended solid (SS) concentrations and the major portion was in solid fractions. After the pretreatment step, they were removed, with 80% for total P, 85% for total Cu, and 84% for total Zn. After the activated sludge process, total P, Cu, and Zn were then removed at 83%, 96%, and 95%, respectively. Removing SS thoroughly at each step was shown to be the most important factor in preventing outflow of these elements, since there are linear correlations or a positive relationship between the removal of SS concentrations and their removal in solid form. Most of the P, Cu, and Zn in activated sludge effluent was in soluble form, and the concentrations of Cu and Zn in the effluent were low enough, while further P removal might be required.

Characteristics of the microbial community associated with ammonia oxidation in a full-scale rockwool biofilter treating malodors from livestock manure composting.

The relationship between the activity and community structure of microbes associated with the oxidation of ammonia in a full-scale rockwool biofilter was examined by kinetic, denaturing gradient gel electrophoresis (DGGE), and sequence analyses. The packing materials were sampled from two different depths at 3 sites. Estimated K(m) values were similar among depths at same sampling sites, while V(max) differed in the mid-point sample. The lower depth of this site had the highest V(max). A correspondence analysis showed the DGGE profile of ammonia-oxidizing bacterial amoA of the lower depth of the mid-point sample to be distinguishable from the others. Banding patterns at other sites were similar among depths. Banding patterns of ammonia-oxidizing archaeal amoA of the mid-point sample were also similar among depths. The results suggested an association between the ammonia-oxidizing bacterial community's composition and ammonium oxidation kinetics in samples. Sequence analysis indicated that the ammonia-oxidizing bacterial community mainly belonged to the Nitrosomonas europaea lineage and Nitrosospira cluster 3. The ammonia-oxidizing archaeal amoA-like sequences were related to those belonging to soil and sediment groups, including one with 84% nucleotide similarity with Nitrosopumilus maritimus.

Rate determination and distribution of anammox activity in activated sludge treating swine wastewater.

This paper presents a quantitative investigation and analysis of anammox activity in sludge taken from biological swine wastewater treatment plants. An incubation experiment using a (15)N tracer technique showed anammox activity in sludge taken from 6 out of 13 plants with the rate ranging from 0.0036 micromol-N(2)/g-VSS/h to 3.1 micromol-N(2)/g-VSS/h, and in a biofilm with the highest activity at 25.8 micromol-N(2)/g-VSS/h. It is notable that 9 out of 11 sludges in which the pH was maintained between 6.6 and 8.1 retained anammox activity, while those with either a lower or higher pH did not. Moreover, anammox-positive sludge had a significantly higher concentration of NO(2)(-)-N plus NO(3)(-)-N than did anammox-negative sludge. A significant difference was not observed between anammox-positive and -negative sludge regarding BOD/NH(4)(+)-N in the influent, DO concentration in aeration tanks, and the concentrations of NH(4)(+)-N, free nitric acid, and free ammonia.

Nitrogen removal by co-occurring methane oxidation, denitrification, aerobic ammonium oxidation, and anammox.

The pathway for removing NO(3)(-) and NH(4)(+) from wastewater in the presence of both CH(4) and O(2) was clarified by studying microbial activity and community. Batch incubation tests were performed to characterize the microbial activity of the sludge, which was acclimatized in a bioreactor in which O(2) and CH(4) were supplied to treat wastewater containing NO(3)(-) and NH(4)(+) . The tests showed that the sludge removed significant amounts of NO(3)(-) and NH(4)(+) in the presence of CH(4) and O(2), and the presence of the activity of methane oxidation, denitrification, nitrification, and anammox in the sludge. It was estimated that the total inorganic nitrogen removal was attributed to denitrification associated with methane oxidation as 53.4%, microbial assimilation as 37.9%, and anammox as 8.7%. Nitrification also contributed to NH(4)(+) decrease as 34.5% and anammox as 6.4%. Anammox activity was unambiguously demonstrated by (29)N(2) production in anaerobic batch incubation with (15)N-labeled inorganic nitrogen compounds. The presence of methane-oxidizing bacteria and candidate denitrifiers in the sludge was shown by denaturing gradient gel electrophoresis of 16S rRNA gene fragments. Clone library analysis of the PCR-amplified 16S rRNA gene fragment using specific primers for aerobic ammonium oxidizer and anammox revealed the presence of these bacteria. The results reveal that complex nitrogen-removal processes occur in the presence of CH(4) and O(2) by methanotroph, denitrifier, aerobic ammonium oxidizer, and anammox.

Continuous hydrogen production from glucose by using extreme thermophilic anaerobic microflora.

Continuous hydrogen production from glucose at 75 degrees C was examined at HRTs of 0.67, 1, 2, and 3 d by using extreme thermophilic microflora, which consisted of several subspecies of Caldanaerobacter subterraneus. The maximum hydrogen yield of 3.32 mol-H2/mol-glucose degraded was obtained at the highest HRT, 3 d. Both the maximum volumetric and specific rates of hydrogen production (1.22 L-H2/L/d and 4.15 L-H2/g-VSS/d, respectively) were obtained at the lowest HRT, 0.67 d. A 16S rDNA analysis showed that the community structure of the microflora did not change at any of the HRTs.

Nitrogen removal from purified swine wastewater using biogas by semi-partitioned reactor.

Nitrate and ammonium removal from purified swine wastewater using biogas and air was investigated in continuous reactor operation. A novel type of reactor, a semi-partitioned reactor (SPR), which enables a biological reaction using methane and oxygen in the water phase and discharges these unused gases separately, was operated with a varying gas supply rate. Successful removal of NO(3)(-) and NH(4)(+) was observed when biogas and air of 1L/min was supplied to an SPR of 9L water phase with a NO(2,3)(-)-N and NH(4)(+)-N removal rate of 0.10 g/L/day and 0.060 g/L/day, respectively. The original biogas contained an average of 77.2% methane, and the discharged biogas from the SPR contained an average of 76.9% of unused methane that was useable for energy like heat or electricity production. Methane was contained in the discharged air from the SPR at an average of 2.1%. When gas supply rates were raised to 2L/min and the nitrogen load was increased, NO(3)(-) concentration was decreased, but NO(2)(-) accumulated in the reactor and the NO(2,3)(-)-N and NH(4)(+)-N removal activity declined. To recover the activity, lowering of the nitrogen load and the gas supply rate was needed. This study shows that the SPR enables nitrogen removal from purified swine wastewater using biogas under limited gas supply condition.