Growth and physiological response of lemongrass (Cymbopogon citratus (D.C.) Stapf.) under different levels of fly ash-amended soil

Revegetation with metal tolerant plants for management of fly ash deposits is an important environmental perspective nowadays. Growth performance, photosynthesis, and antioxidant defense of lemongrass (Cymbopogon citratus (D.C.) Stapf.) were evaluated under various combination of fly ash amended with garden soil in order to assess its fly ash tolerance potential. Under low level of fly ash (25%) amended soil, the plant growth parameters such as shoot, root, and total plant biomass as well as metal tolerance index were increased compared to the control plants grown on garden soil, followed by decline under higher concentration of fly ash (50%, 75% and 100%). In addition, leaf photosynthetic rate, stomatal conductance, and photosystem (PS) II activity were not significantly changed under low level of fly ash (25%) amended soil compared to the garden soil but these parameters were significantly decreased further with increase of fly ash concentrations. Furthermore, increase of activities of some antioxidant enzymes such as superoxide dismutase, ascorbate peroxidase, and guaiacol peroxidase over control were noticed in lemongrass under all fly ash treatments. Taken together, the study suggests that lemongrass can be used for phytoremediation of fly ash at 25% amended soil.     

Effect of feed to inoculum ratios on biogas yields of food and green wastes

Biogas and methane yields of food and green wastes and their mixture were determined using batch anaerobic digesters at mesophilic (35 ± 2 °C) and thermophilic (50 ± 2 °C) temperatures. The mixture was composed of 50% food waste and 50% green waste, based on the volatile solids (VS) initially added to the reactors. The thermophilic digestion tests were performed with four different feed to inoculum (F/I) ratios (i.e., 1.6, 3.1, 4.0 and 5.0) and the mesophilic digestion was conducted at one F/I (3.1). The results showed that the F/I significantly affected the biogas production rate. At four F/Is tested, after 25 days of thermophilic digestion, the biogas yield was determined to be 778, 742, 784 and 396 mL/g VS for food waste, respectively; 631, 529, 524 and 407 mL/g VS for green waste, respectively; and 716, 613, 671 and 555 mL/g VS for the mixture, respectively. About 80% of the biogas production was obtained during the first 10 days of digestion. At the F/I of 3.1, the biogas and methane yields from mesophilic digestion of food waste, green waste and their mixture were lower than the yields obtained at thermophilic temperature. The biogas yields were 430, 372 and 358 mL/g VS, respectively, and the methane yields were 245, 206, and 185 mL/g VS, respectively.     

Suitability of Brahmi (Bacopa monnieri L.) cultivation on fly ash-amended soil for better growth and oil content

Abstract

Sustainable application of fly ash and its management in agriculture is a major challenge nowadays. A pot culture experiment was conducted to find out the most suitable level of fly ash application for soil amendments that can improve the plant growth and productivity of Brahmi (Bacopa monnieri L.). After growing seedlings of B. monnieri under different levels of fly ash for 90?days, a significant increase in plant biomass, essential oil content and tolerance index (more than 100%) was observed under 25% of fly ash amended soil in comparison to garden soil and higher fly ash treatments. Leaf chlorophyll content and photosynthetic parameters were remained unchanged under 25% of fly ash as compared to seedlings grown on garden soil. However, these parameters were significantly declined under higher concentrations of fly ash treatments. Higher levels of fly ash caused oxidative damage and the induction of some antioxidative enzymes activities in B. monnieri indicates its capability to endure oxidative stress tolerance. Overall, our study showed that 25% of fly ash can be used as soil amendment for cultivation of B. monnieri L. leading to enhance plant biomass and essential oil production.     

Nitrogen removal from wastewater in vertical flow constructed wetlands containing LWA/gravel layers and reed vegetation

The influence of light weight aggregates made of fly ash from sewage sludge thermal treatment (FASSTT LWA) on the nitrogen removal efficiency from artificial wastewater in constructed wetlands (CW) with vertical flow reed bed was investigated. Thirty lysimeters with six different double-layer bed constructions (upper layer of FASSTT LWA with thicknesses of: 0 cm, 12 cm, 25 cm, 50 cm, and 100 cm of the total depth of the lysimeter, above a lower gravel layer), either with or without reed plants were operated with wastewater hydraulic loading rate of 4.67 mm/d. During a six-month experiment, high efficiency of ammonia removal was observed. The influence of FASSTT LWA as a bed material and the presence of reed on CW treatment efficiency was determined. The highest total nitrogen removal efficiency, 59.5%, was obtained in the CW with double-layer lysimeters consisting of 25% FASSTT LWA (upper layer), and 75% gravel (lower layer), and planted with reed.     

The performance of biological anaerobic filters packed with sludge-fly ash ceramic particles (SFCP) and commercial ceramic particles (CCP) during the restart period: Effect of the C/N ratios and filter media

Two lab-scale upflow biological anaerobic filters (BAF) packed with sludge-fly ash ceramic particles (SFCP) and commercial ceramic particles (CCP) were employed to investigate effects of the C/N ratios and filter media on the BAF performance during the restart period. The results indicated that BAF could be restarted normally after one-month cease. The C/N ratio of 4.0 was the thresholds of nitrate removal and nitrite accumulation. TN removal and phosphate uptake reached the maximum value at the same C/N ratio of 5.5. Ammonia formation was also found and excreted a negative influence on TN removal, especially when higher C/N ratios were applied. Nutrients were mainly degraded within the height of 25 cm from the bottom. In addition, SFCP, as novel filter media manufactured by wastes–dewatered sludge and fly ash, represented a better potential in inhibiting nitrite accumulation, TN removal and phosphate uptake due to their special characteristics in comparison with CCP.     

Soil properties and turf growth on a sandy soil amended with fly ash

Field lysimeters of a sandy soil were amended to a depth of 100 mm with four rates (0, 5, 10 and 20%, wt/wt) of fly ash, and effects on soil water content, nutrient leaching, turf growth and nutrition, and uptake of trace elements by turf were assessed. Measurements were taken for 70 days for lysimeters either planted with rhizomes of Cynodon dactylon(L.) Pers., cv. `Wintergreen', or left bare. When irrigated daily, soil water content increased progressively with increasing rates of fly ash and leachate volumes were decreased by 17–52% for lysimeters containing fly ash amended soil. Fertiliser was applied equivalent to 28.4 g N m<sup>–2</sup> and 10.3 g P m<sup>–2</sup> for the entire 70 days (including pre-plant application). Macronutrient concentrations in leaf tissue were within levels regarded as sufficient. Total dry mass (root plus shoot) decreased when fertiliser application rates were reduced by 25%, irrespective of fly ash treatment. In `bare' lysimeters containing fly ash amended soil, cumulative leaching of NO₃ ^–, NH₄ ⁺and P were 0.32–0.88 of the values in non-amended soil. When planted with turf, leaching of those nutrients was minimal (equivalent to 3% of total N applied) and leaching loses did not differ among fly ash rates. Extractable soil P levels were increased 2.5–4.5-fold in the fly ash amended zone, compared with non-amended soil. Root mass in the top 100 mm was 1.2–1.5-fold larger for turf in fly ash amended soil, compared to non-amended soil. The Se concentrations were higher in leaf tissue grown in fly ash amended soil (being at most 0.63 g g^–1), but there was no effect of fly ash amended soil on As, Ba, B, Cd, Co, Cr, Cu, Pb, Hg, Mn, Ni, Ag or Zn in leaf tissues. Thus, fly ash amendment may be a suitable management option for turf culture on sandy soils, since fly ash improved soil water holding capacity and root growth in the amended zone.     

AM真菌对煤矿沉陷区不同复合基质上 紫花苜蓿生长和生理特性的影响

该研究采用盆栽试验法,以紫花苜蓿(Medicago sativa)为材料,将煤矸石(CG)、粉煤灰(FA)和沙土(SS)按不同质量配比设置T1(CG∶FA∶SS=75%∶25%∶0%)、T2(CG∶FA∶SS=60%∶25%∶15%)、T3(CG∶FA∶SS=45%∶25%∶30%)、T4(CG∶FA∶SS=30%∶25%∶45%)四组混合基质以模拟,并以T5(CG∶FA∶SS=0%∶0%∶100%)为对照,AM真菌选用摩西斗管囊霉(F.m)和幼套近明球囊霉(C.e),通过单接菌和混合接菌,探索其对紫花苜蓿根系的侵染率、幼苗生长、抗氧化物酶活性和渗透调节物质变化的影响。结果表明:(1)在五组基质上,接种AM真菌显著提高了紫花苜蓿根系的菌根侵染率和菌根依赖性,且基质T4接种F.m+C.e[F.m∶C.e=1∶1(W/W)]的值最大(64.31%和86.24%)。(2)接种AM真菌不同程度提高了紫花苜蓿株高、基径、叶面积和生物量,且混合接菌的效果优于单接菌。(3)基质中填加过量煤矿废弃物抑制了植株根系的生长,接种AM真菌后显著提高了紫花苜蓿总根长、根表面积、根体积,降低了根平均直径。(4)不同接菌处理的紫花苜蓿叶片POD、SOD、CAT活性以及可溶性糖、可溶性蛋白含量总体表现为F.m+C.eC.eF.mnon-AMF,且接种F.m+C.e的增长幅度最大。研究表明煤矿废弃物复合逆境抑制紫花苜蓿的生长,接种AM真菌显著提高了幼苗生长、抗氧化物酶活性和渗透调节物质,提高了植物抗逆性能,且以基质T4接种F.m+C.e的效果最佳。     

Improving pig manure conversion into biogas by thermal and thermo-chemical pretreatments

Thermal (70–190 °C) and thermo-chemical (pH = 10 and 12, 25 °C and 90–190 °C) treatments were investigated in order to maximise the production of methane from pig manure. Methane production from treated and raw manure was assessed from batch mesophilic biochemical methane potential tests. Methane potential of manure soluble fraction increased with the temperature of thermal treatments whereas temperatures higher than 135 °C were necessary to improve the methane potential of the total fraction. The best results were obtained with the highest temperature (190 °C).     

Turf culture under declining volume and frequency of irrigation on a sandy soil amended with fly ash

The effects of four rates (0, 5, 10 and 20%, wt/wt) of fly ash amendment in a sandy soil (top 100–120 mm) on soil properties, turf (Cynodon dactylon (L.) Pers., cv. Wintergreen) water relations, growth and colour, were assessed during 84 days of irrigation treatments (irrigated daily, every 3^rd day, or every 4^th day) imposed during summer in a Mediterranean-type climate. In plots irrigated at 40% of net evaporation summed and applied every 3^rd day: (i) soil water contents were 14–33% higher in the fly ash amended soil zone when compared to values in plots with non-amended soil; (ii) soil water content below the root zone (i.e., 1500 mm) during that period remained low (being only 1–2% above the permanent wilting point), indicating minimal, if any, deep drainage. Extractable soil P was 2.0- to 3.8-fold higher in the fly ash amended soil compared to non-amended soil. By contrast extractable P was 1.7- to 2.1-fold higher in the soil 100–500 mm below the surface in non-amended plots, compared with fly ash amended plots. Irrigation at 40% replacement of net evaporation summed and applied every 3^rd day did not adversely impact on turf growth or colour, when compared to plots irrigated daily, irrespective of fly ash treatments. However, extending irrigations (at 40% of net evaporation) to every 4^th day reduced turf growth and colour, but the turf recovered fully from the mild water stress within 21 days of being irrigated daily at 100% replacement of net evaporation. Therefore, 40% replacement of net evaporation summed and applied every 3^rd day was a suitable watering schedule for maintenance of turf, with minimal risks of deep drainage.     

Effects of fluctuating moisture and temperature regimes on the persistence of quiescent conidia of Isaria fumosorosea

Conidia of Isaria fumosorosea were submitted to three regimes of temperature and moisture to simulate microclimatic conditions which prevail in temperate (43% RH and 28 °C to 98% RH and 15 °C), subtropical (75% RH and 35 °C to 98% RH and 25 °C), and arid areas (13% RH and 40 °C to 33% RH and 15 °C) with daily fluctuating cycles. Germination, conidial viability, and virulence to Spodoptera frugiperda larvae were less affected after 20 days exposure under temperate cycling conditions than under arid and subtropical conditions. Exposure of conidia for 20 days to constant nocturnal simulated conditions of any tested regime weakly affected conidial persistence, whereas diurnal conditions exerted the most detrimental effects of high temperatures. However, when tested at both 45 °C and 50 °C at 33% RH for 160 h, the persistence of I. fumosorosea conidia was relatively higher than expected. These results emphasize that climatic conditions prevailing in environments and ecological fitness of fungal isolates have to be taken into account for assessing microbial control strategies.     

Biosorption of reactive dye by waste biomass of Nostoc linckia

Potential of spent biomass of a cyanobacterium, Nostoc linckia HA 46, from a hydrogen bioreactor was studied for biosorption of a textile dye, reactive red 198. The waste biomass was immobilized in calcium alginate and used for biosorption of the dye from aqueous solution using response surface methodology (RSM). Kinetics of the dye in aqueous solution was studied in batch mode. Interactive effects of initial dye concentration (100-500 mg/L), pH (2-6) and temperature (25-45 °C) on dye removal were examined using Box-Behnken design. Maximum adsorption capacity of the immobilized biomass was 93.5 mg/g at pH 2.0, initial concentration of 100 mg/L and 35 °C temperature, when 94% of the dye was removed. Fourier transform infrared (FT-IR) studies revealed that biosorption was mainly mediated by functional groups like hydroxyl, amide, carboxylate, methyl and methylene groups present on the cell surface.     

Relationships between temperature, development and survival of different life stages of the tomato fruit fly, Neoceratitis cyanescens

The development and survival of female Neoceratitis cyanescens (Bezzi) (Diptera: Tephritidae) from egg to complete ovarian maturation were studied in the laboratory at five different constant temperatures: 15, 20, 25, 30, and 35 °C. The aim of this study was to get information on the influence of temperature on pre-mature stages, as a prerequisite to optimise rearing procedures and to understand temporal and geographical patterns of fruit fly occurrence. The developmental rate of the different life stages increased linearly with increasing temperatures up to 30 °C. The fastest development of pre-mature stages was recorded at 30 °C (22±1 days) and the slowest at 15 °C (98±3 days). The day-degrees requirements (K) to complete total development were 432.6 day-degrees. Lower temperature thresholds were 11.4, 11.9, 10.0, and 11.1 °C for egg, larval, pupal stages and ovarian maturation, respectively. The number of adults obtained from an initial batch of 100 eggs reached a maximum (64) at 25 °C. At 35 °C, no adults emerged. Larval developmental time was significantly shorter in green tomato fruits than in potato tubers at 15, 20, and 25 °C. Mortality rate of larvae was higher in green tomato fruits than in potato tubers at 25 and 30 °C.     

Heat and lime-treatment as effective control methods for E. coli O157:H7 in organic wastes

Land-application of abattoir wastes is economically appealing and may provide an effective means of closing the nutrient cycling loop. This practise is constrained, however, by legislation which necessitates pre-treatment to remove pathogenic micro-organisms prior to land-spreading. Here we investigated whether heat-treatment or lime addition could eliminate Escherichia coli O157:H7 from three contrasting abattoir wastes. We found that treatment at 60 °C for 10 min effectively eradicated the organism while treatment for the same length of time at 50 °C led to 2–4 log reductions, but not a complete kill. Temperatures of 72 °C induced waste solidification rendering its use impractical. The potential for re-growth in heat-treated and untreated wastes was also investigated. Survival was significantly greater in heat-treated wastes, although the difference was less than half a log unit in magnitude. This effect of heat-treatment on pathogen survival appeared to be ameliorated when wastes were mixed with soil. No viable E. coli O157:H7 cells were recovered from any waste after application of lime (CaO) at a rate of 10 g l⁻¹, even after enrichment. Our results indicate that pasteurisation-style or liming treatments may provide a suitable alternative method for reducing pathogen loads in abattoir wastes, so that they can be applied to land with minimal biological risk.     

Continuous production of β-cyclodextrin from starch by highly stable cyclodextrin glycosyltransferase immobilized on chitosan

Cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. was covalently immobilized on glutaraldehyde-activated chitosan spheres and used in a packed bed reactor to investigate the continuous production of β-cyclodextrin (β-CD). The optimum temperatures were 75 °C and 85 °C at pH 6.0, respectively for free and immobilized CGTase, and the optimum pH (5.0) was the same for both at 60 °C. In the reactor, the effects of flow rate and substrate concentration in the β-CD production were evaluated. The optimum substrate concentration was 4% (w/v), maximizing the β-CD production (1.32 g/L) in a flow rate of 3 mL/min. In addition, the biocatalyst had good operational stability at 60 °C, maintaining 61% of its initial activity after 100 cycles of batch and 100% after 100 h of continuous use. These results suggest the possibility of using this immobilized biocatalyst in continuous production of CDs.     

The combined influence of sub-optimal temperature and salinity on the in vitro viability of Perkinsus marinus, a protistan parasite of the eastern oyster Crassostrea virginica

Perkinsus marinus is a major cause of mortality in eastern oysters along the Gulf of Mexico and Atlantic coasts. It is also well documented that temperature and salinity are the primary environmental factors affecting P. marinus viability and proliferation. However, little is known about the effects of combined sub-optimal temperatures and salinities on P. marinus viability. This in vitro study examined those effects by acclimating P. marinus at three salinities (7, 15, 25 ppt) to 10 °C to represent the lowest temperatures generally reached in the Gulf of Mexico, and to 2 °C to represent the lowest temperatures reached along the mid-Atlantic coasts and by measuring changes in cell viability and density on days 1, 30, 60 and 90 following acclimation. Cell viability and density were also measured in 7 ppt cultures acclimated to each temperature and then transferred to 3.5 ppt. The largest decreases in cell viability occurred only with combined low temperature and salinity, indicating that there is clearly a synergistic effect. The largest decreases in cell viability occurred only with both low temperature and salinity after 30 days (3.5 ppt, 2 °C: 0% viability), 60 days (3.5 ppt, 10 °C: 0% viability) and 90 days (7 ppt, 2 °C: 0.6 ± 0.7%; 7 ppt, 10 °C: 0.2 ± 0.2%).     

Coal fly ash and lime addition enhances the rate and efficiency of decomposition of food waste during composting

To evaluate the use of coal fly ash (CFA) on the decomposition efficiency of food waste, synthetic food waste was mixed with lime at 1.5% and 3% (equivalent to 0.94% and 1.88% CaCO₃, respectively), CFA at 5%, 10% and 15% with lime so as to achieve CaCO₃ equivalent of 1.88% and composted for 42 days in a thermophilic 20 l composter with two replicates each. Alkaline materials at 1.88% CaCO₃ equivalent successfully buffered the pH during the composting and enhanced the decomposition efficiency. When these buffering was achieved with CFA + lime, the composting period could be shortened to ∼28 days compared with ∼42 days in 3% lime. Organic decomposition in terms of CO₂ loss, carbon turnover and nitrogen transformation were significantly higher for treatments with 1.88% CaCO₃ equivalent. Nutrient transformations and compost maturity parameters indicated that addition of CFA (5–10%) with lime at 1.88% CaCO₃ equivalent enhances the decomposition efficiency and shortens the composting period by 35%.     

Diapause pupal color diphenism induced by temperature and humidity conditions in Byasa alcinous (Lepidoptera: Papilionidae)

We investigated whether diapause pupae of Byasa alcinous exhibit pupal color diphenism (or polyphenism) similar to the diapause pupal color polyphenism shown by Papilio xuthus. All diapause pupae of B. alcinous observed in the field during winter showed pupal coloration of a dark-brown type. When larvae were reared and allowed to reach pupation under short-day conditions at 18 °C under a 60 ± 5% relative humidity, diapause pupae exhibited pupal color types of brown (33%), light-brown (25%), yellowish-brown (21%), diapause light-yellow (14%) and diapause yellow (7%). When mature larvae reared at 18 °C were transferred and allowed to reach pupation at 10 °C and 25 °C under a 60 ± 5% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 91.2, 8.8 and 0.0% at 10 °C, and 12.2, 48.8 and 39.0% at 25 °C, respectively. On the other hand, when mature larvae reared at 18 °C were transferred and allowed to reach pupation at 10 °C, 18 °C and 25 °C under an over 90% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 79.8, 16.9 and 3.3% at 10 °C, 14.5, 26.9 and 58.6% at 18 °C, and 8.3, 21.2 and 70.5% at 25 °C, respectively. These results indicate that diapause pupae of brown types are induced by lower temperature and humidity conditions, whereas yellow types are induced by higher temperature and humidity conditions. The findings of this study show that diapause pupae of B. alcinous exhibit pupal color diphenism comprising brown and diapause yellow types, and suggest that temperature and humidity experienced after a gut purge are the main factors that affect the diapause pupal coloration of B. alcinous as environmental cues.     

Tolerance and recovery responses of playa halophytes to light,salinity and temperature stresses during seed germination

Halogeton glomeratus (M. Bieb.) C.A. Mey., Lepidium latifolium Linn. and Peganum harmala Linn. are distributed in temperate salt playa habitats of Upper Hunza, Pakistan. Seeds were germinated under various salinity (0–500 mM NaCl), light (12 h-light:12 h-dark and 24 h-dark) and temperature (5/15, 10/20, 15/25, 20/30, and 25/35 °C, dark/light) regimes for 20 days to determine the optimal conditions for germination and recovery of seeds from these factors when exposed to less than optimal conditions. Seeds that failed to germinate in dark were transferred successively to 12 h-photoperiod, salinity to distilled water and from various temperature regimes to 20/30 °C, to determine the effect of these stresses and the ability of these seeds to recover respectively. Highest seed germination (H. glomeratus and L. latifolium: 100%; P. harmala: 80%) was obtained in non-saline control at 20/30 °C in 12 h-photoperiod, however, increase in salinity progressively inhibited seed germination. Seed germination of H. glomeratus and P. harmala was substantially inhibited and that of L. latifolium was prevented in dark. Salinity and dark treatments have a synergistic effect in inhibiting seed germination of all species. No seed of any species germinated at 5/15 °C; germination was substantially inhibited at 25/35 °C both for H. glomeratus and P. harmala while L. latifolium failed to germinate at 25/35 °C. Rate of germination also decreased with an increase in salinity at all temperature regimes but this effect was minimal at optimal temperature regime of 20/30 °C. After successive elimination of light, salinity and temperature stresses, final seed germination was identical to respective controls. The results indicate that seeds of these temperate halophytes could endure environmental stresses without losing viability and germinate readily when these stresses are removed. Under the extremely variable conditions of the playa habitat these species are highly opportunistic exploiting the windows of opportunity available during spring or early summer.     

The effect of soil temperature,moisture and nitrogen on Striga asiatica (L.) Kuntze seed germination,viability and emergence on sorghum (Sorghum bicolor L. Moench) roots under field conditions

Experiments were conducted in a Striga-sick field to study the effect of soil temperature, moisture and nitrogen on Striga parasitism on sorghum. Striga seeds contained in nylon bags and buried at 2 cm in the soil, were exposed to different temperature and moisture treatments. Clear polythene, hay mulch and bare soil treatments were used to vary soil temperature. These treatments gave mean maximum temperatures of 60°, 48° and 37°C, respectively at 2-cm soil depth. Irrigation levels of 0, 30 and 60 mm were applied to change soil moisture. Striga seed germination, viability and emergence were studied. After 34 days of preconditioning, the exhumed Striga seeds from polythene-covered plots (solarized plots) did not germinate or retain viability when these seeds were exposed to sorghum root exudate. However, seeds similarly buried under hay mulch or bare soil, with mean maximum soil temperatures of 48° and 37°C, respectively, had similar germination and viability percentages. Of these 75% germinated and 85% of them were viable, regardless of the temperature treatment. Although seeds stored at high temperature and humidity (solarization) were killed, more Striga plants emerged under the polythene treatment compared to hay mulch and bare soil treatments. The observed Striga plants in the polythene mulch treatment were, therefore, assumed to have come from deeper layers where solarization was not effective. Irrigation treatments did not have significant effects on Striga seed germination and viability, but a slightly higher number of plants emerged at 60-mm irrigation level than at 30-mm and 0-mm. Striga emergence, on the other hand, was directly related to the rate of N application. Nitrogen rates of 0, 25, 50 and 100 kg ha^–1 resulted in the emergence of 11, 34, 38 and 40 Striga plants per plot, respectively. Despite the high infestation at high N levels, sorghum plants did not show a loss of vigor. Nitrogen application, therefore, does not reduce Striga incidence, but seems to neutralize the harmful effects of Striga without reducing the extent of parasitism.