A novel perchlorate- and nitrate-reducing bacterium, Azospira sp. PMJ.

A novel perchlorate-reducing bacterium (PCRB), PMJ, was isolated from the mixed liquor suspended solids in the aerobic tank of a wastewater treatment plant. The 16S ribosomal RNA (rRNA), perchlorate reductase, and chlorite dismutase gene sequences revealed that PMJ belonged to the genus Azospira. PMJ was removed high-strength (700 mg/L) perchlorate and also removed low-strength (≤50 mg/L) perchlorate below the detection limit (2 µg/L) when acetate was used as a sole and carbon source. The maximum specific perchlorate utilization rate, q max, was 0.96 mg ClO4 (-)/mg dry cell weight day, and the half-saturation constant, K S , was lower than 0.002 mg ClO4 (-)/L. PMJ also utilized inorganic electron donors [(H2, S(0), and Fe(II)] with perchlorate as an electron acceptor. Perchlorate reduction by PMJ was completely inhibited by oxygen and chlorate but was not inhibited by nitrate. In the presence of similar concentrations (100∼140 mg/L) of nitrate and perchlorate, PMJ simultaneously removed both electron acceptors. Therefore, it was concluded that the strains PMJ might possess separate pathways for perchlorate and nitrate reduction. These results indicated that Azospira sp. PMJ could be efficiently used for treating perchlorate-contaminated groundwater and wastewater because many of these water bodies are known to contain both perchlorate and nitrate. In addition, low K S value and autotrophic perchlorate reduction of PMJ might be useful to design the biological treatment systems.

A comparative study on the alternating mesophilic and thermophilic two-stage anaerobic digestion of food waste.

An alternating mesophilic and thermophilic two stage anaerobic digestion (AD) process was conducted. The temperature of the acidogenic (A) and methanogenic (M) reactors was controlled as follows: System 1 (S1) mesophilic A-mesophilic M; (S2) mesophilic A-thermophilic M; and (S3) thermophilic A-mesophilic M. Initially, the AD reactor was acclimatized and inoculated with digester sludge. Food waste was added with the soluble chemical oxygen demand (SCOD) concentrations of 41.4-47.0 g/L and volatile fatty acids of 2.0-3.2 g/L. Based on the results, the highest total chemical oxygen demand removal (86.6%) was recorded in S2 while S3 exhibited the highest SCOD removal (96.6%). Comparing S1 with S2, total solids removal increased by 0.5%; S3 on the other hand decreased by 0.1 % as compared to S1. However, volatile solids (VS) removal in S1, S2, and S3 was 78.5%, 81.7%, and 79.2%, respectively. S2 also exhibited the highest CH4 content, yield, and production rate of 70.7%, 0.44 L CH4/g VSadded, and 1.23 L CH4/(L·day), respectively. Bacterial community structure revealed that the richness, diversity, evenness, and dominance of S2 were high except for the archaeal community. The terminal restriction fragments dendrogram also revealed that the microbial community of the acidogenic and methanogenic reactors in S2 was distinct. Therefore, S2 was the best among the systems for the operation of two-stage AD of food waste in terms of CH4 production, nutrient removal, and microbial community structure.

Enhanced butanol production in Clostridium acetobutylicum ATCC 824 by double overexpression of 6-phosphofructokinase and pyruvate kinase genes.

This study elucidated the importance of two critical enzymes in the regulation of butanol production in Clostridium acetobutylicum ATCC 824. Overexpression of both the 6-phosphofructokinase (pfkA) and pyruvate kinase (pykA) genes increased intracellular concentrations of ATP and NADH and also resistance to butanol toxicity. Marked increases of butanol and ethanol production, but not acetone, were also observed in batch fermentation. The butanol and ethanol concentrations were 29.4 and 85.5 % higher, respectively, in the fermentation by double-overexpressed C. acetobutylicum ATCC 824/pfkA+pykA than the wild-type strain. Furthermore, when fed-batch fermentation using glucose was carried out, the butanol and total solvent (acetone, butanol, and ethanol) concentrations reached as high as 19.12 and 28.02 g/L, respectively. The reason for improved butanol formation was attributed to the enhanced NADH and ATP concentrations and increased tolerance to butanol in the double-overexpressed strain.

Ventilation induced evolution pattern of archaea, fungi, bacteria and their potential roles during co-bioevaporation treatment of concentrated landfill leachate and food waste.

To obtain a favorable aeration type in co-bioevaporation treatment of concentrated landfill leachate and food waste, and to deeply understand the co-bioevaporation mechanisms, the temporal evolution differences of archaea, fungi and bacteria as well as the related microbial metabolism genes and functional enzymes under intermittent ventilation (IV) and continuous ventilation (CV) were investigated. Results through metagenomics analysis showed that the less sufficient oxygen and longer thermophilic phase in IV stimulated the vigorous growth of archaea, while CV was beneficial for fungal growth. Even genes of carbohydrates and lipids metabolism and ATP-associated enzymes (enzyme 2.7.13.3 and 3.6.4.12), as well as peptidoglycan biosynthesis enzyme (enzyme 3.4.16.4), were more abundant in CV, IV hold better DNA repair ability, higher microbial viability, and less dehydrogenase sensitivity to temperatures due to the critical contribution of Pseudomonas (3.1-45.9%). Furthermore, IV consumed a similar amount of heat for water evaporation with nearly half of the ventilation of CV and was a favorable aeration type in the practical application of co-bioevaporation.

Struvite production from anaerobic digestate of piggery wastewater using ferronickel slag as a magnesium source.

This study aimed to fully recover ammonia contained at a high concentration in anaerobic digestate of piggery wastewater (ADPW) by forming struvite. As magnesium and phosphorus sources, ferronickel slag (FNS) and K2HPO4 were used, respectively. By leaching 200 g L-1 of FNS with 3.0 M H2SO4, 10,309 mg L-1 of magnesium ions were extracted, and this acid-leachate of FNS (FNSL) also contained 5965 mg L-1 of total iron. In order to simultaneously remove both high concentrations of organic matters in ADPW and iron in FNSL which were known to hinder struvite formation, the mixture of ADPW and FNSL was added with H2O2 at the H2O2/Fe molar ratio of 0.75 and pH 4.0. After Fenton reaction, removal efficiencies of COD and total iron reached 77.36% and 99.89%, respectively. Then COD and an iron-reduced mixture of ADPW and FNSL were added with K2HPO4 satisfying Mg:N:P molar ratio of 1.2:1:1.15 at pH 9.5 to produce struvite for 1 h. From 1 L of ADPW (2.21 g NH3-N), 0.65 L of FNSL (4.65 g Mg2+), and 5.63 g of PO4 3-P, 46.7 g of precipitates were obtained. Overall removal efficiencies of magnesium, NH3-N, and phosphorus were 98.59%, 94.25%, and 99.97%, respectively. Obtained precipitates were analysed by using XRD, XRF, SEM-EDX and found to be struvite with impurities of potassium and metals. Additionally, the economic feasibility of FNS was assessed by estimating chemical costs of various magnesium sources.

Microbial community response to ciprofloxacin toxicity in sponge membrane bioreactor.

This study aims to offer insights into how ciprofloxacin (CIP) impact bacterial community structures in the Sponge-MBR process when CIP is spiked into hospital wastewater. We found that the CIP toxicity decreased richness critical phylotypes such as phylum class ẟ-, ß-, É£-proteobacteria, and Flavobacteria that co-respond to suppress denitrification and cake fouling to 37% and 28% respectively. Cluster analysis shows that the different community structures were formed under the influence of CIP toxicity. CIP decreased attached growth biomass by 2.3 times while increasing the concentration of permeate nitrate by 3.8 times, greatly affecting TN removal by up to 26%. Ammonia removal was kept stable by inflating the ammonia removal rate (p < 0.003), with the wealthy Nitrospira genus guaranteeing the nitrification activity. In addition, we observed an increasing richness of Chloroflexi and Planctomycetes, which may play a role in fouling reduction in the Sponge-MBR. Therefore, if the amount of antibiotics in hospital wastewater continues to increase, it is so important to extend biomass retention for denitrification recovery.

Effect of ciprofloxacin dosages on the performance of sponge membrane bioreactor treating hospital wastewater.

This study aimed to evaluate treatment performance and membrane fouling of a lab-scale Sponge-MBR under the added ciprofloxacin (CIP) dosages (20; 50; 100 and 200 µg L-1) treating hospital wastewater. The results showed that Sponge-MBR exhibited effective removal of COD (94-98%) during the operation period despite increment of CIP concentrations from 20 to 200 µg L-1. The applied CIP dosage of 200 µg L-1 caused an inhibition of microorganisms in sponges, i.e. significant reduction of the attached biomass and a decrease in the size of suspended flocs. Moreover, this led to deteriorating the denitrification rate to 3-12% compared to 35% at the other lower CIP dosages. Importantly, Sponge-MBR reinforced the stability of CIP removal at various added CIP dosages (permeate of below 13 µg L-1). Additionally, the fouling rate at CIP dosage of 200 µg L-1 was 30.6 times lower compared to the control condition (no added CIP dosage).

Electrochemical DNA biosensors based on thin gold films sputtered on capacitive nanoporous niobium oxide.

Electrochemical DNA biosensors based on a thin gold film sputtered on anodic porous niobium oxide (Au@Nb(2)O(5)) are studied in detail here. We found that the novel DNA biosensor based on Au@Nb(2)O(5) is superior to those based on the bulk gold electrode or niobium oxide electrode. For example, the novel method does not require any time-consuming cleaning step in order to obtain reproducible results. The adhesion of gold films on the substrate is very stable during electrochemical biosensing, when the thin gold films are deposited on anodically prepared nanoporous niobium oxide. In particular, the novel biosensor shows enhanced biosensing performance with a 2.4 times higher resolution and a three times higher sensitivity. The signal enhancement is in part attributed to capacitive interface between gold films and nanoporous niobium oxide, where charges are accumulated during the anodic and cathodic scanning, and is in part ascribed to the structural stability of DNA immobilized at the sputtered gold films. The method allows for the detection of single-base mismatch DNA as well as for the discrimination of mismatch positions.

Protein recovery from excess sludge for its use as animal feed.

In this study, the possibility of using proteins recovered from excess sludge as animal feed was investigated. The proteins were recovered through the processes of sludge disintegration (alkali treatment followed by ultra-sonication), precipitation and drying of the soluble proteins. The compositions and the toxicants of the recovered proteins were analyzed, and the toxicity was assessed by Sprague-Dawley (SD) rat experiments. The results showed that the nutrient compositions were comparable with the commercial protein feeds. Heavy metals were found to be removed after the protein recovery process, and aflatoxin B1, ochratoxin A and Salmonella D groups were not detected. The rat toxicity tests showed that there were no effects on mortality, the incidence of clinical signs, body weight changes, and necropsy findings. The minimum lethal dose (MLD) was higher than 2000 mg/kg. Based on these results, the use of the crude protein recovered from excess sludge as animal feed appears to be technically feasible.

Reuse of low concentrated electronic wastewater using selected microbe immobilised cell system.

This work describes a novel technology for the reuse of low concentrated electronic wastewater using selected microbe immobilisation cell (SMIC) system. The SMIC system is an innovative technology to maximise the activity of specific microorganisms capable of decomposing tetramethyl ammonium hydroxide (TMAH) as a major organic compound in the low concentrated electronic wastewater. The versatility of the SMIC system has been studied by using continuous-flow reactors. The TOC in a SMIC system was removed completely, indicating that SMIC is a useful technology to remove TOC biologically in low concentrated wastewater. The most important advantages of this system are highly effective and stable in view of TMAH removal. These characteristics make well suited to various applications depending on targeted compounds and microorganisms and, especially, in the wastewater of electronic facilities.

Spent coffee ground as a new bulking agent for accelerated biodrying of dewatered sludge.

The feasibility of using spent coffee ground (SCG) as a new bulking agent for biodrying of dewatered sludge (DS) was investigated in comparison with two other frequently-used bulking agents, air-dried sludge (AS) and sawdust (SD). Results showed that the moisture contents (MC) of 16-day DS biodrying with AS (Trial A), SCG (Trial B) and SD (Trial C) decreased from 70.14 wt%, 68.25 wt% and 71.63 wt% to 59.12 wt%, 41.35 wt% and 57.69 wt%, respectively. In case of Trial B, the MC rapidly decreased to 46.16 wt% with the highest water removal (70.87%) within 8 days because of the longest high-temperature period (5.8 days). Further studies indicated that the abundant biodegradable volatile solids (BVS) and high dissolved organic matter (DOM) contents in SCG were the main driving forces for water removal. According to pyrosequencing data, Firmicutes, most of which were recognized as thermophiles, was rapidly enriched on Day 8 and became the dominant phylum in Trial B. Four thermophilic genera, Bacillus, Ureibacillus, Geobacillus and Thermobifida, which can produce thermostable hydrolytic extracellular enzymes, were the most abundant in Trial B, indicating that these thermophilic bacteria evolved during the long high-temperature period enhanced the biodegradation of BVS in SCG. The 8-day biodried product of Trial B was demonstrated to be an excellent solid fuel with low heating value (LHV) of 9284 kJ kg-1, which was 2.1 and 1.8 times those of biodried products with AS and SD, respectively. Thus SCG was found to be an excellent bulking agent accelerating DS biodrying and producing a solid fuel with a high calorific value.

Combustion characteristics of biodried sewage sludge.

In this study, effects of biodrying on the characteristics of sewage sludge and the subsequent combustion behavior were investigated. 7-Day of biodrying removed 49.78% of water and 23.17% of VS initially contained in the sewage sludge and increased lower heating value (LHV) by 37.87%. Meanwhile, mass contents of C and N decreased from 36.25% and 6.12% to 32.06% and 4.82%, respectively. Surface of the biodried sewage sludge (BDSS) appeared granulated and multi-porous, which was thought to facilitate air transfer during combustion. According to thermogravimetric (TG) analysis coupled with mass spectrometer (MS) with a heating rate of 10 °C/min from 35 °C to 1000 °C, thermally-dried sewage sludge (TDSS) and BDSS lost 74.39% and 67.04% of the initial mass, respectively. In addition, combustibility index (S) of BDSS (8.67 × 10-8 min-2 K-3) was higher than TDSS. TG-MS analyses also showed that less nitrogenous gases were generated from BDSS than TDSS. It was again showed that the average CO and NO concentrations in exit gas from isothermal combustion of BDSS were lower than those from TDSS, especially at low temperatures (≤800 °C). Based on these results, it was concluded that biodrying of sewage sludge was an energy-efficient water-removal method with less emission of air pollutants when BDSS was combusted.

Recovery of ammonia through struvite production using anaerobic digestate of piggery wastewater and leachate of sewage sludge ash.

Anaerobic digestate of piggery wastewater (ADPW) contains high concentrations of ammonia and phosphorus with unbalanced molar ratio. Thus, ammonia remains at a high level even after phosphorus is completely removed through struvite formation. In this study, both ammonia and phosphorus were recovered by adding leachate of sewage sludge ash (SSA) into ADPW. It was demonstrated that 11,600 mg L-1 of total phosphorus and 7266.7 mg L-1 of [Formula: see text]-P were extracted from SSA by using sulfuric acid at the H2SO4/SSA mass ratio of 0.35. ADPW and the leachate of SSA were mixed at the volumetric ratio of 1:1.29, and then struvite was formed at the molar ratio of 1.2 (Mg2+):1.0 ([Formula: see text]-P):1.0 (NH3-N). Removal efficiencies of ammonia and phosphorus were 91.95% and 99.65%, respectively. The obtained struvite was analyzed by various methods and was found to meet the Korean fertilizer standards, except for copper.

Effect of 3,3',4',5-tetrachlorosalicylanilide on reduction of excess sludge and nitrogen removal in biological wastewater treatment process.

A metabolic uncoupler, 3,3',4',5-tetrachlorosalicylanilide (TCS), was used to reduce excess sludge production in biological wastewater treatment processes. Batch experiments confirmed that 0.4 mg/l of TCS reduced the aerobic growth yield of activated sludge by over 60%. However, the growth yield remained virtually constant even at the increased concentrations of TCS when cultivations were carried out under the anoxic condition. Reduction of sludge production yield was confirmed in a laboratory-scale anoxic-oxic process operated for 6 months. However, it was found that ammonia oxidation efficiency was reduced by as much as 77% in the presence of 0.8 mg/l of TCS in the batch culture. Similar results were also obtained through batch inhibition tests with activated sludges and by bioluminescence assays using a recombinant Nitrosomonas europaea (pMJ217). Because of this inhibitory effect of TCS on nitrification, the TCS-fed continuous system failed to remove ammonia in the influent. When TCS feeding was stopped, the nitrification yield of the process was resumed. Therefore, it seems to be necessary to assess the nitrogen content of wastewater if TCS is used for reducing sludge generation.

Structural and Kinetic Characteristics of 1,4-Dioxane-Degrading Bacterial Consortia Containing the Phylum TM7.

1,4-Dioxane-degrading bacterial consortia were enriched from forest soil (FS) and activated sludge (AS) using a defined medium containing 1,4-dioxane as the sole carbon source. These two enrichments cultures appeared to have inducible tetrahydrofuran/dioxane and propane degradation enzymes. According to qPCR results on the 16S rRNA and soluble di-iron monooxygenase genes, the relative abundances of 1,4-dioxane-degrading bacteria to total bacteria in FS and AS were 29.4% and 57.8%, respectively. For FS, the cell growth yields (Y), maximum specific degradation rate (Vmax), and half-saturation concentration (Km) were 0.58 mg-protein/mg-dioxane, 0.037 mg-dioxane/mg-protein∙h, and 93.9 mg/l, respectively. For AS, Y, Vmax, and Km were 0.34 mg-protein/mg-dioxane, 0.078 mg-dioxane/mg-protein∙h, and 181.3 mg/l, respectively. These kinetics data of FS and AS were similar to previously reported values. Based on bacterial community analysis on 16S rRNA gene sequences of the two enrichment cultures, the FS consortium was identified to contain 38.3% of Mycobacterium and 10.6% of Afipia, similar to previously reported literature. Meanwhile, 49.5% of the AS consortium belonged to the candidate division TM7, which has never been reported to be involved in 1,4-dioxane biodegradation. However, recent studies suggested that TM7 bacteria were associated with degradation of non-biodegradable and hazardous materials. Therefore, our results showed that previously unknown 1,4-dioxane-degrading bacteria might play an important role in enriched AS. Although the metabolic capability and ecophysiological significance of the predominant TM7 bacteria in AS enrichment culture remain unclear, our data reveal hidden characteristics of the TM7 phylum and provide a perspective for studying this previously uncultured phylotype.

A rapid and simple respirometric biosensor with immobilized cells of Nitrosomonas europaea for detecting inhibitors of ammonia oxidation.

As obligate chemolithotrophs, ammonia-oxidizing bacteria (AOB) grow very slowly and are known to be extremely sensitive to a wide variety of inhibitors. Since it is generally accepted that inhibition of ammonia oxidation by AOB results in a total failure of nitrogen removal, it is necessary to develop a method to detect inhibitors of ammonia oxidation in wastewater. Since ammonia oxidation accompanies oxygen consumption, ammonia oxidation can be easily evaluated by measuring oxygen consumption rate using a dissolved oxygen (DO) probe. In this study, a rapid and simple respirometric biosensor using the pure culture of Nitrosomonas europaea was developed. N. europaea was cultivated in a continuous fermentor operating at the dilution rate of 0.008 h(-1) to obtain physiologically constant cells and was immobilized onto the dialysis membrane through filtration. DO, determined by the biosensor, started to increase 30 s later after ammonia oxidation inhibitor was fed, and a new steady-state DO was obtained in 10-30 min. For this DO profile, steady-state kinetics was applied to evaluate ammonia oxidation efficiency. The concentration of a toxic compound causing 50% decrease of oxygen-consumption activity (EC50) was determined for different chemicals. The EC50 values obtained with the biosensor (0.018 mg l(-1) for allylthiourea, 0.027 mg l(-1) for thioacetamide, 1.10 mg l(-1) for phenol and 0.0 1mg l(-1) for thiourea) indicated that the developed biosensor was highly sensitive to a variety of the inhibitors. It was also shown that the biosensor is applicable for on-line real time monitoring.

Purification and characterization of NADPH-dependent Cr(VI) reductase from Escherichia coli ATCC 33456.

A soluble Cr(VI) reductase was purified from the cytoplasm of Escherichia coli ATCC 33456. The molecular mass was estimated to be 84 and 42 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, indicating a dimeric structure. The pI was 4.66, and optimal enzyme activity was obtained at pH 6.5 and 37 degrees C. The most stable condition existed at pH 7.0. The purified enzyme used both NADPH and NADH as electron donors for Cr(VI) reduction, while NADPH was the better, conferring 61%; higher activity than NADH. The Km values for NADPH and NADH were determined to be 47.5 and 17.2 micromol, and the Vmax values 322.2 and 130.7 micromol Cr(VI) min(-1)mg(-1) protein, respectively. The activity was strongly inhibited by N-ethylmalemide, Ag2+, Cd2+, Hg2+, and Zn2+. The antibody against the enzyme showed no immunological cross reaction with those of other Cr(VI) reducing strains.

A novel continuous toxicity test system using a luminously modified freshwater bacterium.

An automated continuous toxicity test system was developed using a recombinant bioluminescent freshwater bacterium. The groundwater-borne bacterium, Janthinobacterium lividum YH9-RC, was modified with luxAB and optimized for toxicity tests using different kinds of organic carbon compounds and heavy metals. luxAB-marked YH9-RC cells were much more sensitive (average 7.3-8.6 times) to chemicals used for toxicity detection than marine Vibrio fischeri cells used in the Microtox assay. Toxicity tests for wastewater samples using the YH9-RC-based toxicity assay showed that EC50-5 min values in an untreated raw wastewater sample (23.9 +/- 12.8%) were the lowest, while those in an effluent sample (76.7 +/- 14.9%) were the highest. Lyophilization conditions were optimized in 384-multiwell plates containing bioluminescent bacteria that were pre-incubated for 15 min in 0.16 M of trehalose prior to freeze-drying, increasing the recovery of bioluminescence and viability by 50%. Luminously modified cells exposed to continuous phenol or wastewater stream showed a rapid decrease in bioluminescence, which fell below detectable range within 1 min. An advanced toxicity test system, featuring automated real-time toxicity monitoring and alerting functions, was designed and finely tuned. This novel continuous toxicity test system can be used for real-time biomonitoring of water toxicity, and can potentially be used as a biological early warning system.

Nitrogen control in AO process with recirculation of solubilized excess sludge.

In order to establish a sludgeless process with a nitrogen-controlled effluent, batch and continuous experiments in a lab scale anoxic-oxic (AO) process were carried out to investigate the possibility of ozonized sludge (OS) usage as a denitrification energy source. Through ozonation at an ozone dose of 1.2g O(3)/g MLVSS, 63.2% of treated MLVSS was solubilized, 12.7% of COD was lost (probably due to complete oxidation to CO(2)), and soluble COD/TN ratio of OS appeared to be only about 10.78 because ozonation released cellular proteins and other nitrogenous substances. In oxic conditions, incubation of OS supernatant with activated sludge generated nitrate without significant ammonia accumulation, which meant that rapid nitrification occurred following ammonia generation from heterotrophic degradation of nitrogen-bearing cellular substances. In anoxic conditions, externally supplied nitrate was removed at the expense of organic carbons in the OS supernatant. However, ammonia was accumulated as anoxic incubation proceeded probably because of heterotrophic degradation of nitrogenous materials as in oxic conditions. Thus it was appeared that solubilized excess sludge acted as a reducing power for denitrification but also as a nitrogen source. In addition, 24-41% of COD contained in OS supernatant were found to be consumed for denitrification. But the remaining COD was not assimilated further even in the presence of nitrate. It was concluded by a nitrogen balance analysis that the energy source contained in OS was not sufficient to completely reduce the nitrogen that was originated from OS itself to nitrogen gas.

Novel bioevaporation process for the zero-discharge treatment of highly concentrated organic wastewater.

A novel process termed as bioevaporation was established to completely evaporate wastewater by metabolic heat released from the aerobic microbial degradation of the organic matters contained in the highly concentrated organic wastewater itself. By adding the glucose solution and ground food waste (FW) into the biodried sludge bed, the activity of the microorganisms in the biodried sludge was stimulated and the water in the glucose solution and FW was evaporated. As the biodegradable volatile solids (BVS) concentration in wastewater increased, more heat was produced and the water removal ratio increased. When the volatile solids (VS) concentrations of both glucose and ground FW were 120 g L(-1), 101.7% and 104.3% of the added water was removed, respectively, by completely consuming the glucose and FW BVS. Therefore, the complete removal of water and biodegradable organic contents was achieved simultaneously in the bioevaporation process, which accomplished zero-discharge treatment of highly concentrated organic wastewater.