Electron donors and acceptors influence anaerobic soil organic matter mineralization in tidal marshes

Anaerobic decomposition in wetland soils is carried out by several interacting microbial processes that influence carbon storage and greenhouse gas emissions. To understand the role of wetlands in the global carbon cycle, it is critical to understand how differences in both electron donor (i.e., organic carbon) and terminal electron acceptor (TEA) availability influence anaerobic mineralization of soil organic matter. In this study we manipulated electron donors and acceptors to examine how these factors influence total rates of carbon mineralization and the pathways of microbial respiration (e.g., sulfate reduction versus methanogenesis). Using a field-based reciprocal transplant of soils from brackish and freshwater tidal marshes, in conjunction with laboratory amendments of TEAs, we examined how rates of organic carbon mineralization changed when soils with different carbon contents were exposed to different TEAs. Total mineralization (the sum of CO₂ + CH₄ produced) on a per gram soil basis was greater in the brackish marsh soils, which had higher soil organic matter content; however, on a per gram carbon basis, mineralization was greater in the freshwater soils, suggesting that the quality of carbon inputs from the freshwater plants was higher. Overall anaerobic metabolism was higher for both soil types incubated at the brackish site where SO₄²⁻ was the dominant TEA. When soils were amended with TEAs in the laboratory, more thermodynamically favorable respiration pathways typically resulted in greater organic matter mineralization (Fe(III) respiration > SO₄²⁻ reduction > methanogenesis). These results suggest that both electron donors and acceptors play important roles in regulating anaerobic microbial mineralization of soil organic matter.     

A model describing the interactions between anaerobic microbiology and geochemistry in a soil amended with glucose and nitrate

Under anaerobic conditions, microbes closely interact with geochemical reactions and can have an impact on the soil, the deep vadose zone, the underlying aquifer and the atmosphere. We have designed a model combining anaerobic microbial activities with geochemical reactions in the soil, and assessed it in batch experiments. The model describes the dynamics of six functional microbial communities, their decomposition after death, and the catabolism of carbohydrates through denitrification, dissimilatory NH₄⁺ production, Fe(III) reduction, fermentation, acetogenesis, and SO₄^2– reduction. It was combined with a model that thermodynamically describes acid–base, reduction–oxidation and complexation reactions in solution, and kinetic precipitation and dissolution. Batch incubations were done on a Calcic Cambisol, either without amendment, or after supplying (i) glucose or (ii) glucose and NO₃^–. Gases, mineral cations and anions, glucose, fatty acids and alcohols were measured during incubation. Net production of CO₂ was similar for both glucose treatments, about 40 times larger than in the control. For the glucose treatments, the main microbial activities were fermentation, acetogenic transformation of ethanol, and oxidation of H₂. When the soil was enriched with NO₃^–, no H₂ was produced, and microbial activities were rapidly inhibited by NO₂^–. The model shows these trends as well as geochemical characteristics including pH and reduction–oxidation potential.     

湿地甲烷厌氧氧化机制研究进展

微生物主导的甲烷厌氧氧化（AOM）一直是甲烷（CH₄）氧化研究的前沿热点问题,对控制全球湿地CH4排放具有重要意义。介绍了硫酸盐还原型（SAMO）、反硝化型（DAMO）、金属氧化物驱动型（Metal-AOM）和中间电子直接转移型（DIET）四种AOM途径,其中,SAMO以SO₄^2-作为AOM的最终电子受体,DAMO以 NO₂^-/ NO₃^-作为AOM的最终电子受体,Metal-AOM以铁锰等金属离子/金属氧化物作AOM的最终电子受体,而种间电子转移是一种不需要中间产物的转移方式。详细阐述了途径中主要功能微生物菌群的相关研究,重点讨论了AOM途径中可能发生的反应机理和进展,论述了目前常用的微生物群落结构和丰度检测分析的分子生物学方法。最后,从湿地环境因子对参与AOM微生物的影响机制、功能微生物富集培养以及湿地微生物参与环境污染控制等方面进行展望。这对于进一步研究全球湿地碳、氮循环提供了微生物学方面的参考。     

Anaerobic reoxidation of Mn2+, Fe2+, S0 and S2– in submerged paddy soils

Anaerobic reoxidation of reduced products in paddy soils was investigated. Ferrous iron (Fe²⁺) and monosulfide ion (S^2–) added to the soil chemically reduced MnO₂ to Mn²⁺, and MnO₂ and Fe(OH)₃ to Mn²⁺ and Fe²⁺, respectively, where Fe²⁺ and S^2– were considered to be oxidized to Fe³⁺ and S⁰. Elemental sulfur was oxidized to sulfate by anaerobic incubation with NO₃ ^– MnO₂ and Fe(OH)₃. A new conceptual model for the reduction processes in submerged paddy soil including the reoxidation processes of reduced products, in which soil heterogeneity in paddy fields was taken into consideration, was proposed based on the results. Received: 20 October 1996     

Nitrous oxide production in a sequence batch reactor wastewater treatment system using synthetic wastewater

The rate of nitrous oxide emission from a laboratory sequence batch reactor （SBR） wastewater treatment system using synthetic wastewater was measured under controlled conditions. The SBR was operated in the mode of 4 h for aeration, 3.5 h for stirring without aeration, 0.5 h for settling and drainage, and 4 h of idle. The sludge was acclimated by running the system to achieve a stable running state as chemical oxygen demand, NO2^-, NO3^-, NH4^＋, pH, and N2O. indicated by rhythmic changes of total N, dissolved oxygen, Under the present experimental conditions measured nitrous oxide emitted from the off-gas in the aerobic and anaerobic phases, respectively, accounted for 8.6%-16.1% and 0-0.05% of N removed, indicating that the aerobic phase was the main source of N2O emission from the system. N2O dissolved in discharged water was considerable in term of concentration. Thus, measures to be developed for the purpose of reducing N2O emission from the system should be effective in the aeration phase.     

Effects of nitrite toxicity on soil bacteria under aerobic and anaerobic conditions

Isolates of a soil Pseudoimonas, as well as other soil bacteria, showed a different sensitivity towards NO^?₂ when grown under aerobic or anaerobic conditions. The tolerance to NO^?₂ was increased in the presence of O₂: for instance, a concentration of $\text{200parts}\text{10}{}^6$ of NO^?₂-N proved to be toxic to a Pseudomonas sp. under anaerobic conditions, whereas over $\text{400 parts}\text{10}{}^6$ were needed aerobically to suppress its growth completely. The addition of NO^?₃ as an electron acceptor for anaerobic respiration did not overcome the inhibitive effect of NO^?₃. The pH range, at which NO^?₂ was utilized anaerobically, was narrowed with increasing NO^?₂ concentration (pH 6.8–8.8 at $\text{70 parts}\text{10}{}^6$ of NO^?₂-N and 7.4–8.5 and $\text{140 parts}\text{10}{}^6$ of NO^?₂-N).Tolerance to nitrite varied considerably among the bacteria tested. Each species was able to overcome the inhibitory effect of NO^?₂ up to a certain concentration, while the length of the lag phase was related to NO^?₂ concentration.     

旱作褐土中铁氧化物的厌氧还原动力学特征

异化铁还原是厌氧环境中有机物降解的重要微生物过程,不仅影响Cr、U等无机污染物在环境中的迁移,还与CH4、N2O、H2S等温室气体的释放关系密切.本文选择7个旱作褐土样品,采用泥浆厌氧恒温培养的方法,研究了旱作褐土中氧化铁的厌氧还原特征,结果表明旱作褐土在淹水条件下可以发生铁氧化物的异化还原,其还原潜势介于4.47～5.95 mg g-1之间,还原速率常数介于0.15 ～0.27 d-1之间.褐土中部分晶态铁氧化物可在这一过程中被还原,游离铁的平均还原率为41.95％.经过40 d厌氧培养后,99.26％的NO3-和88.82％的SO42-被还原.还原过程的速率常数κ、Vmax、Tmax随着土壤有机碳含量增加而增加,还原潜势与土壤SO42-含量呈显著负相关关系.     

Increase in Cation Adsorption Induced by Surface Complexation of Sulfate on Andisols and Prediction by “Four-Plane Model”

Abstract

The “Four-plane model” is one of the surface complexation models developed for analyzing the electrostatic charge of synthetic oxides. This model which was applied to the B horizon of Andisols, was equilibrated with an electrolyte solution containing KCl, KNO₃, K₂SO₄ in the concentration range of 0.0033, 0.01, 0.033, 0.1 M, and pH range of 2 to 11. The amounts of NO₃ ^-, Cl^-, SO₄ ^2-, and K⁺ adsorbed were determined from the products remaining in the liquid phase. The increase in the pH value results in the increase of K⁺ adsorption, and decrease of anion adsorption. The increase in the equilibrium concentration increased both K⁺ and anion adsorption. The parameters of the model were determined by measurements and the iterative method. The model have a good agreement between calculated and measured values of cation and anion adsorption. Among the anion species, the amount of adsorbed anions was in the order of SO₄ ^2-?Cl^->NO₃ ^-. The differences in the degree of adsorption were mainly associated with the differences in the equilibrium constants of surface complexation. The surface complexation of the anions produced a negative charge and increased cation adsorption. The degree of surface complexation was most significant in the K₂SO₄ solution, and the phenomenon was considered to be important not only for anion adsorption but also for cation adsorption on Andisols.     

Characteristics of SO2, NO2, Soot and Benzo(a)Pyrene Concentration Variation on the Eastern Coast of the Baltic Sea

The investigation of SO₂, NO₂, soot and benzo(a)pyrene (BP) has been performed at the background station on the eastern coast of the Baltic Sea since 1980. A significant decreasing trend has been observed for SO₂ and NO₂, while soot and BP concentrations were changing insignificantly. The decreasing SO₂ and NO₂ high concentrations (>10µg·m^?3) have been determined in the air masses coming from the Western and Central Europe to Lithuania since 1900. The concentration of SO₂ in a range of 0–5µg·m^?3 and the concentration of NO₂ in a range of 0–10µg·m^?3 are characteric of the background atmospheric air.     

Development of a Multi-Resolution Emission Inventory and Its Impact on Sulfur Distribution for Northeast Asia

Emissions in East Asia for 1993 by administrative units and source types are estimated to support regional emission assessments and transport modeling studies. Total emission of SO_x, NO_x, soil NO_x, N₂O, and NH₃ are 24 150, 12 610, 1963, 908, and 8263 kton yr^-1, respectively.China's emission contribution is the highest for every species.The area sources are the most significant source type for SO_x and NO_x, but the fraction due to mobile source is highest for NO_x. Major LPSs are located from the middle to the east part of China, south and middle-west part of South Korea, and the east part of Japan. The area sources of SO_x show a pattern similar to population density, whereas NH₃ shows a strong landuse dependency. Detail emissions analysis reveals higher SO_x emission `cores' within each province. The estimated emissions are used to estimate sulfur deposition in the regions. The seasonal average sulfur distribution amounts are estimated from the ATMOS2 chemical transport model. The results showed anti-correlation with temperature for sulfur (SO₂ + SO₄ ^-2) concentrations and a positive correlation with rainfall for deposition.     

A regional scale model for the calculation of episodic concentrations and depositions of acidifying components

An eulerian long-range transport model for the calculation of concentrations of SO₂, SO₄, NO _x , and NO₃ and wet and dry depositions of SO _x (sum of SO₂ and SO₄) and NO _y (sum of NO, NO₂ and NO₃) over Europe is presented. The model is developed in such a way that only routinely available, analyzed or prognostic meteorological fields are required as input data. In this way it is possible to obtain a forecast of the air quality during smog episodes. For evaluation of smog episodes the model provides a way to estimate the contributions of different sources and the effect of emission scenarios. The model has been evaluated for four winter and three summer episodes. The modeled concentrations of SO₂ and SO, are in agreement with the available measurements. A less good agreement is found for NO₂ and NO _x (sum of NO and NO₂) concentrations. For these components the model tends to underpredict the measured values.     

Vulnerability Assessments of Colorado Ground Water to Nitrate Contamination

Nitrate (NO₃-N) contamination of ground water aquifers is an important problem in the United States and throughout the world, particularly as ground water resources become increasingly relied upon to support human needs. Cost effective methodologies are needed to facilitate decision-making for ground water protection. To aid ground water protection organizations, we designed two tools to assess aquifer vulnerability to NO₃-N contamination in Colorado. The first tool is a statewide aquifer vulnerability map (VM) that identifies regions vulnerable to ground water contamination. The VM uses five factors that influence aquifer vulnerability on a regional scale: aquifer locations, depth to water, soil drainage class, land use, and recharge availability. We validated the VM using 576 discrete ground water sample points from throughout the state and found that the VM was able to delineate areas of increased aquifer vulnerability to NO₃-N contamination (r ²= 0.78). The second aquifer assessment tool is a vulnerability matrix (VMX) developed to help practitioners determine relative aquifer vulnerability to NO₃-N contamination on a field scale. The VMX consists of a series of factors that are rated and combined for a particular field. This rating is used to give landowners an index of general aquifer vulnerability to NO₃-N contamination for a specific field, and inform them of changes in management practices to reduce the vulnerability. The VMX can be used in conjunction with the VM to determine NO₃-N contamination potential from intensive agriculture.     

Short-term partitioning of <Superscript>14</Superscript>C-[U]-glucose in the soil microbial pool under varied aeration status

The effect of soil aeration status on carbon partitioning of a labelled organic substrate (¹⁴C-[U]-glucose) into CO₂, microbial biomass, and extra-cellular metabolites is described. The soil was incubated in a continuous flow incubation apparatus under four different aeration conditions: (1) permanently aerobic, (2) permanently anaerobic, (3) shifted from anaerobic to aerobic, and (4) shifted from aerobic to anaerobic. The soil was pre-incubated for 10 days either under aerobic or under anaerobic conditions. Afterwards, glucose was added (315 g C g^–1) and the soils were incubated for 72 h according to four treatments: aerobic or anaerobic conditions maintained, aerobic conditions shifted to anaerobic conditions and anaerobic conditions shifted to aerobic conditions. Carbon partitioning was measured 0, 8, 16, 24, 48 and 72 h after the glucose addition. In permanently aerobic conditions, the largest part of the consumed glucose was built into microbial biomass (72%), much less was mineralised to CO₂ (27%), and only a negligible portion was transformed to soluble extra-cellular metabolites. Microbial metabolism was strongly inhibited when aeration conditions were changed from aerobic to anaerobic, with only about 35% of the added glucose consumed during the incubation. The consumed glucose was transformed proportionally to microbial biomass and CO₂. In permanently anaerobic conditions, 42% of the consumed glucose was transformed into microbial biomass, 30% to CO₂, and 28% to extra-cellular metabolites. After a shift of anaerobic to aerobic conditions, microbial metabolism was not suppressed and the consumed glucose was transformed mainly to microbial biomass (75%) and CO₂ (23%). Concomitant mineralisation of soil organic carbon was always lower in anaerobic than in aerobic conditions.     

Nitrogen Removal and N<Subscript>2</Subscript>O Emission During Low Carbon Wastewater Treatment Using the Multiple A/O Process

With the organic carbon of acetate (SBR-A) and propionate (SBR-P), the effect of organic carbon sources on nitrogen removal and nitrous oxide (N₂O) emission in the multiple anoxic and aerobic process was investigated. The nitrogen removal percentages in SBR-A and SBR-P reactor were both 72%, and the phosphate removal percentages were 97 and 85.4%, respectively. During nitrification, both the NH₄ ⁺-N oxidation rate in the SBR-A and SBR-P had a small change without the influence of the addition of nitrite nitrogen (NO₂ ^?-N). With the addition of 10 mg/L NO₂ ^?-N, the nitrate nitrogen (NO₃ ^?-N) production rate, N₂O accumulation rate and emission factor had increased. At the same time, the N₂O emission factor of SBR-A and SBR-P reactors increased from 2.13 and 0.87% to 4.66 and 2.08%, respectively. During exogenous denitrification, when nitrite was used as electron acceptor, the N₂O emission factors were 34.1 and 8.6 times more than those of NO₃ ^?-N as electron acceptor in SBR-A and SBR-P. During endogenous denitrification with NO₂ ^?-N as electron acceptor, the accumulation rate and emission factor of N₂O were higher than those of NO₃ ^?-N as electron acceptor. High-throughput sequencing test showed that the dominant bacteria were Proteobacteria and Bacteroidetes in both reactors at the phylum level, while the main denitrification functional bacteria were Thauera sp., Zoogloea sp. and Dechloromonas sp. at the genus level.     

Effects of simulated throughfall pH and sulfate concentration on a deciduous forest soil

A model deciduous forest soil (Schaffenaker loamy sand) was treated for 8 mo in the greenhouse in 25 cm reconstructed columns with simulated throughfall at pH 6.0 or 4.0, and SO₄ ^2? levels of 12.8 or 24.8 mg L^?1. Red oak seedlings grown in the microcosms showed no growth or foliar element response to the treatments. Sulfate loading had a greater impact on soil and leachate chemistry than pH. Higher available soil P in the A, horizon was associated with the pH 6.0 and high SO₄ ^2?2 treatment combination. High SO₄ ^2? loading also reduced exchangeable K⁺ in the A_1?. Other soil horizons were unaffected by either treatment. Leachate chemistry was not significantly altered by througfall pH, but significantly greater export of Na⁺, Ca²⁺, Mg²⁺, Al³⁺, and NO₃ ^?, and lower SO₄ ^2? loss, occurred with low SO₄ ^? input. Comparatively half as much NO₃ ^? loss was associated with high SO₄ ^2? deposition. The high rate of NO₃ ^? leaching appeared responsible for greater equivalent mass loss of cations from the low SO₄ ^2? treatment. Leachate removal of SO₄ ^2? approximated input after 8 mo. The capacity of this soil to adsorb SO₄ ^2? appeared relatively limited in the absence of normal element cycling. The sulfate component of simulated deciduous forest throughfall was shown to have a potentially greater impact than pH on ion leaching from forest soil. Additional consideration of the role of SO^2? ₄ deposition, in the context of throughfall rather than incident precipitation, is warranted in studies of acidic deposition effects on internal forest soil processes.     

Study on Dry Deposition of SO2-NOX onto Loess

In order to obtain information on dry deposition of SO₂ onto loess in China, the effects of water vapor and NO₂ on SO₂ deposition-oxidation processes were investigated in laboratory measurements. The deposition velocities of SO₂ onto loess particles were 2.34–7.33 cm s^?1, were high in comparison with the other studies. Deposition of SO₂ onto the particles was decreased with exposed time and amounts of SO₂, but was influenced by adsorption of water vapor onto the particles. On the other hand, oxidation of SO₂ physically8 adsorbed was promoted by the coexistence of water vapor and NO₂. So, it was indicated that it was possible for water vapor and NO₂ in the atmosphere to play an important role in dry deposition for the deposition-oxidation interaction between SO₂ and loess particles.     

Chloride interference in total nitrogen analysis by the Kjeldahl method

Abstract

Very low recovery of NH₄+‐N was observed in total N determination of (NH₄)₂SO₄ in KC1 solutions by a semimicro Kjeldahl method using permanganate and reduced iron to recover NO₃‐ and NO₂‐, whereas complete recovery was obtained in analysis of NH₄+‐N in water, and of NO₃ ^?‐N or NO₂ ^?‐N in either water or KC1 solutions. The loss of NH₄ ⁺‐N observed with KC1 was attributed to the formation of NCl₃ upon reaction of NH₄ ⁺ with Cl₂ generated during oxidation of Cl^? by MnO₄ ^?. This difficulty is avoided by using K₂SO₄ instead of KC1 for extraction of inorganic N from soil. Complete recovery was obtained by adding ¹⁵N‐labeled NH₄+, NO₃‐, or NO₂‐ to 0.5 M K₂SO₄ soil extracts, and total ¹⁵N analyses of the labeled extracts were in good agreement with values calculated from the additions of ¹⁵N and the total N contents of the soil extracts.     

Evalution of Acid Deposition in Korea

This study was carried out to evaluate acid depositions and to understand their effect. Wet precipitation has been collected at twenty-four sites in Korea for one year of 1999. The ion concentrations such as H⁺, Na⁺, K⁺, Mg²⁺, NH₄ ⁺, Ca²⁺, Cl^?, NO₃ ^? and SO₄ ^2? were chemically analyzed and determined. Precipitation had wide range of pH(3.5～8.5), and volume-weighted average was 5.2. The contribution amounts of Cl^?, SO₄ ^2? and NO₃ ^? in anion were shown to be 54%, 32%, and 14%, respectively and those of Na⁺ and NH₄ ⁺ in cation were 32% and 25%. The ratios of Cl^? and Mg²⁺ to Na⁺ in precipitation were similar to those of seawater, which imply that great amount of Cl^? and Mg²⁺ in precipitation could be originated from seawater. The concentration of H⁺ is little related with SO₄ ^2?, NO₃ ^? and Cl^? ions, whereas nss^?SO₄ ^2? and NO₃ ^? are highly correlated with NH₄ ⁺, which could suggest that great amount of SO₄ ^2? and NO₃ ^? exist in the form of ammonium associated salt. The annual wet deposition amounts (g m^?2year^?1) of SO₄ ^2?, NO₃ ^?, Cl^?, H⁺, NH₄ ⁺, Na⁺, K⁺, Ca²⁺ and Mg²⁺ were estimated as 0.88～4.89, 0.49～4.37, 0.30～9.80, 0.001～0.031, 0.06～2.15, 0.27～4.27, 0.10～3.81, 0.23～1.59 and 0.03～0.63.     

Effects of sulfate amendments on mineralization and phosphorus release from South Florida (USA) wetland soils under anaerobic conditions

We investigated the potential effects of elevated water-column sulfate (SO₄) levels on heterotrophic microbial respiration and net phosphorus (P) release for soils collected from impacted and unimpacted Everglades wetlands in south Florida. Soils from three sites, ranging from low P and low SO₄ to high P and high SO₄ environments, were examined under controlled laboratory conditions. The soils were subjected to anaerobic incubations to evaluate net P release and organic matter decomposition in response to SO₄ amendments of 32 or 96 mg l⁻¹ (0.33 and 1.0 mM).Three processes have been described in the literature to explain why SO₄ enrichment may lead to P release from soils under anaerobic conditions. First, alkalinization can lead to a more favorable pH environment for decomposition. For the soils examined here, alkalinization due to the hydrogen ion-consuming reaction of SO₄ reduction was not a prominent mechanism. We found that pH decreased in the incubation vessels, and that increases in alkalinity were more likely attributable to calcium carbonate dissolution than SO₄ reduction. Moreover, all the soils exhibited near circum-neutral pH levels, with moderate to high concentrations of native alkalinity.Second, formation of iron sulfide (FeS_x) compounds has been shown to mobilize iron (Fe)-associated P. Soils from only one of the study sites had Fe concentrations that would be expected to be high enough to influence P mobility. Relatively high porewater Fe:soluble reactive P (SRP) ratios (>83:1) were observed at this site, which suggests that Fe could theoretically exert control over the release of P from the soil. However, soil P levels at this site were too low to measure any substantial influence of Fe on net P mobilization.Finally, availability of electron acceptors such as SO₄ is a major determinant of decomposition rate, and thus rate of organic P release. Amending the soils with SO₄ did not result in either more heterotrophic microbial respiration as measured by carbon dioxide (CO₂) and methane (CH₄) production, or increased net P mobilization. In two of the SO₄-amended soils where post-incubation total sulfide concentrations were as high as 23.4 mg l⁻¹, SO₄ addition reduced production of respiratory carbon end products, suggesting hydrogen sulfide inhibition. Moreover, limitations imposed by substrate quality and low P contributed to the lack of meaningful enhanced decomposition of organic matter with the addition of 32 or 96 mg SO₄ l⁻¹ to the oligotrophic wetland soils. Even though P release did occur under anaerobic conditions for the more enriched site, addition of SO₄ did not enhance P release.     

Ion elution and release sequence from deep snowpacks in the central Sierra Nevada,California

Elution of Cl^?, SO₄ ^2?, NO₃ ^?, and H⁺ often occurred in that order at a site in the central Sierra Nevada, California, that receives an annual average of 1000 cm of snowfall which is low in acidic components. During eight winter periods of above-base level snowpack outflow, and one spring melt period, on the average 25% of the ions were discharged at the following percentages of outflow volumes: Cl^? at 11%, NO₃ ^? at 13%, SO₄ ^2? at 18%, and H⁺ at 20%. Seven of eight winter outflows were associated with low ionic strength rainfall onto the snowpack. Mean solute concentrations during the first 25% and first 50% of the total outflow were significantly greater than during the last 75% and last 50% of the total outflow for Cl^?, NO₃ ^?, and SO₄ ^2?, but not for H⁺. Maximum solute concentrations were up to 2.9 times the overall event volume-weighted mean concentrations for Cl^?, 3.7 times for NO₃ ^?, 3.0 times for SO₄ ^2?, and 2.9 times for H⁺.