Microbial electrolysis cell simultaneously enhancing methanization and reducing hydrogen sulfide production in anaerobic digestion of sewage sludge.

The effects of microbial electrolysis cells (MECs) at three applied voltages (0.8, 1.3, and 1.6 V) on simultaneously enhancing methanization and reducing hydrogen sulfide (H2S) production in the anaerobic digestion (AD) of sewage sludge were studied. The results showed that the MECs at 1.3 V and 1.6 V simultaneously enhanced the methane production by 57.02 and 12.70% and organic matter removal by 38.77 and 11.13%, and reduced H2S production by 94.8 and 98.2%, respectively. MECs at 1.3 V and 1.6 V created a micro-aerobic conditions for the digesters with oxidation-reduction potential as -178∼-232 mv, which enhanced methanization and reduced H2S production. Sulfur reduction, H2S and elemental sulfur oxidation occurred simultaneously in the ADs at 1.3 V and 1.6 V. The relative abundances of sulfur-oxidizing bacteria increased from 0.11% to 0.42% and those of sulfur-reducing bacteria decreased from 1.24% to 0.33% when the applied voltage of MEC increased from 0 V to 1.6 V. Hydrogen produced by electrolysis enhanced the abundance of Methanobacterium and changed the methanogenesis pathway.

Performance enhancement of integrating microbial electrolysis cell on two-stage anaerobic digestion of food waste: Electro-methanogenic stage versus electro-two stages.

The effects of microbial electrolysis cell (MEC) integration stage on two-stage anaerobic digestion (TSAD) of food waste (FW) were studied via semi-continuous experiments. The results showed that both MEC (with 1.2 V) integrations enhanced the performances of the TSADs, with the enhancement of electro-two stages being higher. The methane production of TSAD increased from 1.36 ± 0.04 L/L/d to 1.53 ± 0.05 L/L/d (electro-methanogenic stage) and 1.54 ± 0.04 L/L/d (electro-two stages) during the steady period. Electro-acidogenesis decreased propionic acid production and enhanced hydrogen production, while electro-methanogenesis promoted the conversion of volatile fatty acids to methane. The MEC integration improved energy recovery from the organic matter in FW by 11.65-16.15% and reduced the mass loss, with those of the electro-two stages being higher and the electro-methanogenic stage being dominant in the electro-two stages. The integration of MEC enhanced anaerobic fermentation by enriching Olsenella, norank_f__ST-12K33 and Proteiniphilum and improved methanogenesis by enriching Methanobacterium and Candidatus_Methanofastidiosum.

Improving sewage sludge dewaterability via heterogeneous activation of persulfate by Fe-Al layered double hydroxide: Role of generated SO4-•.

In this study, Fe-Al layered double hydroxide (Fe-Al LDH) was prepared and applied to activate persulfate to condition sewage sludge and improve its dewaterability. The results showed that Fe-Al LDH activated persulfate to generate a large amount of free radicals, which attacked extracellular polymeric substances and reduced their content, disrupted microbial cells, released bound water, decreased sludge particle size, increased sludge zeta potential, and improved sludge dewaterability. After sewage sludge was conditioned with Fe-Al LDH (0.20 g/g total solids (TS)) and persulfate (0.10 g/g TS) for 30 min, the capillary suction time of the sludge dropped from 52.0 s to 16.3 s, while the moisture content of the sludge cake decreased from 93.2% to 68.5%. The dominant active free radical produced by the Fe-Al LDH-activated persulfate was SO4-•. The maximum Fe3+ leaching of the conditioned sludge was only 102.67 ± 4.45 mg/L, thus effectively alleviating the secondary pollution of Fe3+. The leaching rate of 2.37% was significantly lower than that of the sludge homogeneously activated with Fe2+ (738.4 ± 26.07 mg/L and 71.00%).

Effects of temperature and total solid content on biohydrogen production from dark fermentation of rice straw: Performance and microbial community characteristics.

Semi-continuous experiments were carried out in lab-scale continuous stirred tank reactors to evaluate the effects of fermentation temperature (37 ± 1 °C and 55 ± 1 °C) and total solids (TS) contents (3 %, 6 %, and 12 %) on biohydrogen production from the dark fermentations (DF) of rice straw (RS) and the total operation duration was 105 days. The experimental results show that biohydrogen production (0.46-63.60 mL/g VSadded) from the thermophilic (55 ± 1 °C) DF (TDF) was higher than the mesophilic (37 ± 1 °C) DF (MDF) (0.19-2.13 mL/g VSadded) at the three TS contents, and achieved the highest of 63.60 ± 2.98 mL/g VSadded at TS = 6 % in TDF. The pH, NH4+-N and total volatile fatty acid of fermentation liquids in the TDF were all higher than those in the MDF. The high abundance of lactic acid-producing bacteria resulted in low biohydrogen produced at TS = 3 %. Under the TDF with TS = 6 %, the highest abundance of hydrolytic bacteria (Ruminiclostridium 54.24 %) led to the highest biohydrogen production. The increase of TS content from 6 % to 12 % induced degradation pathway changes from biohydrogen production to methane production. This study demonstrated that butyric acid fermentation was the main pathway to produce biohydrogen from RS in both DFs.

Effect of mixing ratio and total solids content on temperature-phased anaerobic codigestion of rice straw and pig manure: Biohythane production and microbial structure.

Long-term semi-continuous experiments were carried out under three feedstock conditions to study the effects of mixing ratio and total solids (TS) content on temperature-phased anaerobic codigestion of rice straw (RS) and pig manure (PM). The results showed that biohythane only produced from the mixture with 6% TS content and its average content were 12.83 ± 1.19% (hydrogen) and 23.68 ± 1.12% (methane). Increasing mixture TS content and decreasing its RS ratio increased biohythane production and organic matter removal by creating a suitable process pH and increasing the anaerobic reaction rates. The highest biohythane production of the mixture reached 73.09 ± 3.03 ml/g VS (hydrogen) and 235.81 ± 9.30 ml/g VS (methane) at a mixing ratio of 5:1 and TS content of 6%. A variety of hydrogen-producing bacteria were found in the thermophilic reactor and Clostridium_sensu_stricto_1 played an important role. Butyric acid fermentation is the main hydrogen-producing pathway. Methanobacterium and Methanosaeta were dominant archaea in the mesophilic reactor.

Effects of substrate organic composition on mesophilic and thermophilic anaerobic co-digestion of food waste and paper waste.

Facing the huge output of food waste (FW) and paper waste (PW), long-term semi-continuous experiments were carried out to investigate the effect of the substrate organic composition on mesophilic and thermophilic anaerobic co-digestions (Co-ADs) of their mixtures. The experimental results showed that the organic composition of the substrate affected the biogas and methane production and yield of the two Co-ADs of the FW and PW mixtures, and its effect on thermophilic Co-AD (Co-TAD) was lower than that on mesophilic Co-AD (Co-MAD). The two Co-ADs had similar biogas (2.158 ± 0.136 L/L/d and 2.183 ± 0.142 L/L/d) and methane production (1.245 ± 0.082 L/L/d and 1.279 ± 0.088 L/L/d), and organic matter degradation (81.79 ± 1.07% and 83.81 ± 1.09%) when the substrate organic composition was carbohydrates:proteins:lipids = 6.8:1.8:1 (low carbohydrate composition, FW:PW = 4:1). When the substrate organic composition was carbohydrates:proteins:lipids = 13.5:2:1 (high carbohydrate composition, FW:PW = 1:1), the thermophilic temperature was more favorable than the mesophilic temperature for the Co-AD of FW and PW mixtures. The characteristics (pH, total ammonia, total volatile fatty acids, and total alkalinity) of the Co-TAD digestate were more sensitive to changes in the organic composition of the substrate than those of the Co-MAD digestate. Increasing the carbohydrate content of the FW:PW mixture lowered the production of biogas and methane, and degradation of organic matter in both Co-ADs.

Effects of low- and high-temperature thermal-alkaline pretreatments on anaerobic digestion of waste activated sludge.

To compare the effects of low- and high-temperature thermal-alkaline pretreatments (LTTAP, 60 ± 1 °C, pH 12.0 ± 0.1, 30 min and HTTAP, 160 ± 1 °C, pH 12.0 ± 0.1, 30 min, respectively) on anaerobic digestion (AD) of waste activated sludge, long-term and semi-continuous experiments were conducted in three laboratory continuous stirred tank reactors. The experimental results showed that the two pretreatments increased the methane yield of sludge from 89.20 ± 2.41 mL/g added volatile solids (VS) to 117.50 ± 5.27 mL/g added VS (LTTAP) and 156.40 ± 2.99 mL/g added VS (HTTAP). After AD, the reduction of sludge (volatile solid) increased from 32.91 ± 0.27% to 44.17 ± 1.53% (LTTAP), and 50.86 ± 1.18% (HTTAP), and the abundance of pathogenic bacteria decreased from 6.53% to 0.38% (LTTAP) and 0.14% (HTTAP). LTTAP enhanced both hydrogentrophic and acetoclastic methanogenis and HTTAP only enhanced acetoclastic methanogenis. Additionally, the energy efficiency of HTTAP and its subsequent AD was lower than that of LTTAP and its subsequent AD.

Improving two-stage thermophilic-mesophilic anaerobic co-digestion of swine manure and rice straw by digestate recirculation.

The anaerobic co-digestion (coAD) of swine manure (SM) and rice straw (RS) is appealing for renewable energy recovery and waste treatment worldwidely. Improving its performance is very important for its application. In this study, long-term semi-continuous experiments were conducted to evaluate the improving effects of digestate recirculation on the performance, energy recovery, and microbial community of two-stage thermophilic-mesophilic coAD of swine manure (SM) and rice straw (RS). The experimental results indicated that the coAD systems of SM and RS (mixing ratio of 3:1) with or without digestate recirculation could not realize phase separation. The reactors of both coAD systems were characterized by pH values ranging from 7.74 to 7.85, methane production as 0.41 ± 0.02 and 0.44 ± 0.03 L/L/d, and stable operation. Notably, digestate recirculation increased total methane production, organic matter removal, and reaction rate of the coAD system by 9.92 ± 5.08, 5.22 ± 1.94, and 9.73-12.60%, respectively. Digestate recirculation improved the performance of the coAD by significantly increasing the abundance of Methanosarcina (from 4.1% to 7.5%-10.7% and 35.7%) and decreasing that of Methanothermobacter (from 94.2% to 87.3%-83.6% and 56.8%). Thus, the main methanogenesis pathway of the coAD system was changed by digestate recirculation and the methane production was effectively improved. Although the energy input of the coAD system increased by 30.26%, digestate recirculation improved the energy balance of the total system by 6.83%.

Bioenergy recovery from methanogenic co-digestion of food waste and sewage sludge by a high-solid anaerobic membrane bioreactor (AnMBR): mass balance and energy potential.

To support smart city in terms of municipal waste management and bioenergy recovery, a high-solid anaerobic membrane bioreactor (AnMBR) was developed for sewage sludge (SeS) and food waste (FW) treatment in this study. COD mass balance showed that 54.1%, 66.9%, 73.5%, 91.4% and 93.5% of the COD input was converted into methane at the FW ratio of 0, 25%, 50%, 75% and 100%, respectively. The corresponding net energy balance was 13.6, 14.1, 17.1, 22.9 and 27.4 kJ/g-VS, respectively. An important finding of this investigation was that, for the first time, the relationship between net energy balance and carbon to nitrogen (C/N) ratio was revealed and the established sigmoid-type function was shown to be capable of predicting energy balance at different C/N ratios regardless of the region. The outcomes of this study show the potential of high-solid AnMBRs in SeS and FW treatment for supporting smart cities in the future.

Dark co-fermentation of rice straw and pig manure for biohydrogen production: effects of different inoculum pretreatments and substrate mixing ratio.

Biohydrogen produced from agricultural waste through dark co-fermentation is an increasingly valuable source of renewable energy. Rice straw (RS) and pig manure (PM) are widely available waste products in Asia with complementary levels of carbon and nitrogen that together have a high biohydrogen production potential. However, no research has yet determined the ideal inoculum pretreatment method and mixing ratio for biohydrogen production using these resources. In this study, we tested biohydrogen production using three different inoculum pretreatment methods (acid, alkali and thermal) at five RS/PM ratios (1:0, 5:1, 3:1, 1:1 and 0:1, based on total solids). All three pretreatments promoted biohydrogen production with the increase of bioactivity of biohydrogen-producing organisms (compared with a control group), though acid was clearly superior to thermal or alkali. Using acid pretreatment and RS/PM ratio of 5:1 corresponded with a relatively low NH4+-N concentration (655.17 mg/L), a maximal cumulative biohydrogen production of 44.59 mL/g VSadded with a low methane production (<0.1%), a large butyric acid accumulation (1035.30 mg/L) and a biohydrogen conversion rate of 2.12%. The optimal pH for biohydrogen production from co-fermentation of RS and PM ranged from 5.0-5.5.

Biohythane production and microbial characteristics of two alternating mesophilic and thermophilic two-stage anaerobic co-digesters fed with rice straw and pig manure.

To investigate biohythane production and microbial behavior during temperature-phased (TP) anaerobic co-digestion (AcD) of rice straw (RS) and pig manure (PM), a mesophilic-thermophilic (M1-T1) AcD system and a thermophilic-mesophilic (T2-M2) AcD system were continuously operated for 95 days in parallel. The maximal ratio (8.44%v/v) of produced hydrogen to methane demonstrated the feasibility of biohythane production by co-digestion of RS and PM. T2-M2 exhibited higher hydrogen (16.68 ± 1.88 mL/gVS) and methane (197.73 ± 11.77 mL/gVS) yields than M1-T1 (3.08 ± 0.39 and 109.03 ± 4.97 mL/gVS, respectively). Methanobrevibacter (75.62%, a hydrogenotrophic methanogen) dominated in the M1 reactor, resulting in low hydrogen production. Hydrogen-producing bacteria (Thermoanaerobacterium 32.06% and Clostridium_sensu_stricto_1 27.33%) dominated in T2, but the abundance of hydrolytic bacteria was low, indicating that hydrolysis could be a rate-limiting step. The thermophilic acid-producing phase provided effective selective pressure for hydrogen-consuming microbes, and the high diversity of microbes in M2 implied a more efficient pathway of methane production.

Comparison and application of biofilter and suspended bioreactor in removing gaseous o-xylene.

Two bioreactors, suspended-growth bioreactors (SPB) and biofilter (BF), were compared for the performances in removing gaseous o-xylene. Their efficiencies were investigated by varying the o-xylene loadings, gas flow rates, and gas-water ratios. High-throughput techniques were applied for the microbial populations assay. The conversion rate of carbon in o-xylene was calculated, and the relationship between biomass and removal efficiencies was also analyzed. Results indicated that both the SPB and BF could effectively treat gases containing o-xylene. The average removal efficiencies were 91.8% and 93.5%, respectively. The elimination capacity of the BF was much higher than that of the SPB when the intake load was below 150 g m-3 h-1. When the o-xylene loadings were over 150 g m-3 h-1, both the SPB and BF achieved similar o-xylene removal rates. The maximum elimination capacities were 28.36 g m-3 h-1 for the SPB and 30.67 g m-3 h-1 for BF. The SPB was more sensitive to the changes in the gas flow rate. Results of microbial assay indicated that bacteria e.g. Mycobacterium sp. and Rhodanobacter sp. might play important roles in removing o-xylene in the SPB, while the bacteria Pseudomonas sp., Sphingomonas sp., and Defluviicoccus sp., and the fungi Aspergillus sp. and Scedosporium sp., were the o-xylene degraders in the BF. The successful application of the integrated bioreactor in treating gases containing o-xylene exhausted from the electroplating plant indicated that the integration of SPB and BF could be an effective method for removing VOCs with Henry coefficient in the range of 0.01-1.

Temperature-phased anaerobic co-digestion of food waste and paper waste with and without recirculation: Biogas production and microbial structure.

Two temperature-phased anaerobic digestion (TPAD) systems (55 °C in the first reactor and 35 °C in the second reactor) with and without recirculation were operated in parallel for the co-digestion of food waste and paper waste. A long-term experiment was carried out for these two systems with the paper waste ratios elevated from 0 to 50%. The removal efficiencies of COD, TS, VS, carbohydrate and protein in the recirculated TPAD system were higher than those of the non-recirculated system. The successful acclimation of thermophilic cellulose-degrading bacteria in the first reactor (RT1), partly due to recirculation, ensured the effective degradation of cellulose when the paper waste ratio was higher than 40%, resulting in the production of large amounts of hydrogen in reactor RT1. In the absence of recirculation, the main substance produced in the first reactor of the non-recirculated system (T1) was lactic acid. This gradually led to over-acidification and a low degradation efficiency and no methane or hydrogen was produced in T1. Recirculation helped to establish a stable bacterial community capable of producing bio-hydrogen in reactor RT1. The relatively low pH of 5.5 in the RT1 inhibited the activity of hydrogenotrophic archaea without consuming hydrogen, facilitating high hydrogen production levels.

Comparison of two advanced anaerobic digestions of sewage sludge with high-temperature thermal pretreatment and low-temperature thermal-alkaline pretreatment.

Semi-continuous experiments were conducted to compare the performances and energy efficiencies of two advanced anaerobic digestions (AAD) of sewage sludge with high-temperature thermal pretreatment (HTTP, 160 ± 1 °C and 0.55 MPa for 30 min) and low-temperature thermal-alkaline pretreatment (LTTAP, 60 ± 1 °C and pH 12.0 ± 0.1 for 30 min), which had similar sludge disintegration degree (9.44-9.48%). At the steady period of a SRT 20 d, the two AAD had similar methane production (150.22 ± 9.55 ml/L/d and 151.02 ± 12.56 ml/L/d) and organic matter removals (22.54 ± 2.84% and 23.15 ± 2.46% for volatile solids-VS). The results of high-throughput sequencing showed that the methanogenic pathways of the two AAD were strictly hydrogenotrophic (AAD with HTTP) and hydrogenotrophic/acetoclastic methanogenesis (AAD with LTTAP), respectively. The energy balance analysis suggested that the AAD with LTTAP was superior to that with HTTP because the former had a higher energy efficiency (1.610) than the latter (1.358).

Performance and microbial community variations of a upflow anaerobic sludge blanket (UASB) reactor for treating monosodium glutamate wastewater: Effects of organic loading rate.

To investigate the effect of the organic loading rate (OLR) on anaerobic treatment of monosodium glutamate (MSG) wastewater, a lab-scale up-flow anaerobic blanket (UASB) reactor was continuously operated over a 222-day period. The overall performances of COD removal and methane recovery initially exhibited an increase and subsequently decreased when the OLR was increased from 1 g-COD/L/d to 24 g-COD/L/d. At the optimal OLR of 8 g-COD/L/d, superior performance was obtained with a maximum COD removal efficiency of 97%, a methane production rate of 2.3 L/L/d, and specific methanogenic activity of 86 mg-CH4/g-VSS/d (feeding on glutamate), with superior characteristics of sludge in VSS concentration, average diameter of granules, and settling velocity. According to the results of the specific methanogenic activity, the methanogenic pathway was more inclined to pass through acetate than through hydrogen. Methanosarcina instead of Methanosaeta, with Methanobacterium and greatly increased Firmicutes, dominated in the UASB reactor after long term operation. These results support that the OLR had a substantial effect on both the treatment and energy recovery efficiency of MSG wastewater as well as on microbial community variations in the UASB reactor.

Anaerobic treatment of glutamate-rich wastewater in a continuous UASB reactor: Effect of hydraulic retention time and methanogenic degradation pathway.

To investigate the anaerobic treatment efficiency and degradation pathways of glutamate-rich wastewater under various hydraulic retention times (HRTs), a lab-scale upflow anaerobic sludge blanket (UASB) reactor was operated continuously for 180 days. Results showed that high chemical oxygen demand (COD) removal efficiencies of 95.5%-96.5% were achieved at HRTs of 4.5 h-6 h with a maximum methane yield of 0.31 L-CH4/g-COD. When the HRT was shortened to less than 3 h, the removal performance of the reactor declined. There also was an excessive accumulation of volatile fatty acids, which implies that an appropriately small HRT is applicable to the UASB reactor treating glutamate-rich wastewater. Methanogenic degradation of glutamate in the UASB reactor depended on the HRT applied, and the typical methane-producing capability of the sludge at an HRT of 3 h, in descending order, was acetate > glutamate > butyrate > H2/CO2 > valerate > propionate. Clostridium and Methanosaeta were predominant in the glutamate-degrading sludge. At least three degradation pathways most likely existed in the UASB reactor, and the pathway via 3-methlaspartate by Clostridium pascui was expected to be dominant.

Evaluation of the secondary structures of protein in the extracellular polymeric substances extracted from activated sludge by different methods.

The changes of protein secondary structures in the extracellular polymeric substances (EPS) extracted from activated sludge by four different methods were studied by analyzing the amide I region (1700-1600 cm-1) of the Fourier transform infrared spectra and model protein test. The results showed the molecular weight distribution of organic matter extracted by centrifugation, heating and cation exchange resin (CER) was similar, while the EPS extracted by centrifugation (Control) and CER had similar fluorescent organic matter. The protein secondary structures of extracted EPS by the four methods were different. The similarities of protein secondary structures between the EPS extracted by CER with the Control were the highest among the four extracted EPS. Although the EPS yield extracted by formaldehyde + NaOH method were the highest, its protein secondary structures had the lowest similarity with those extracted by the Control. Additionally, the effects of centrifugation and CER extraction on the secondary structures of bovine serum albumin were also lower than that of other extraction processes. CER enables the second maximum extraction of EPS and maximum retention of the original secondary structure of proteins.

New insights into the effect of thermal treatment on sludge dewaterability.

The changes of the sludge dewaterability in different thermal treatments and the factors influencing these changes were examined in this study. The experimental results showed that the sludge dewaterability deteriorated by the thermal pretreatment with temperature range from 20 to 170 °C, but the deterioration decreased above a certain temperature threshold (120-150 °C). The factors which affected the dewaterability of thermal-treated sludge in two temperature ranges (20-105 °C and 105-170 °C) were different. The dewaterability of thermal-treated sludge was influenced by the protein, humic acid, and polysaccharide contents of different extracellular polymeric substance fractions and the molecular distribution and fluorescence intensity of tightly bound extracellular polymeric substance in the range from 20 to 105 °C. From 105 to 170 °C, while, the thermal-treated sludge dewaterability was influenced mainly by the α-helix of protein in soluble extracellular polymeric substance. These experimental results provide a new insight into the effect of thermal treatment on sludge dewaterability, which will help guide subsequent research.

Effects of tetrakis (hydroxymethyl) phosphonium sulfate pretreatment on characteristics of sewage sludge.

Recently, tetrakis(hydroxymethyl)phosphonium sulfate (THPS) was found to play an important role in the sludge pretreatment process. However, the effects of THPS pretreatment on the characteristics of sewage sludge are still insufficiently understood. The properties of sludge after pretreatment with different concentrations of THPS were investigated in this study. The results showed that pH, dewatering ability, and particle size of sludge decreased with increase in THPS concentration. The volatile suspended solids (VSS) and total suspended solids (TSS) of sludge also decreased slightly with increase in THPS concentration. The specific oxygen uptake rate (SOUR) results suggested that lower THPS concentrations (≤1.87 mg/g VSS) enhanced the activity of sludge, but higher concentrations (≥1.87 mg/g VSS) inhibited it. Gram-negative bacteria with peritrichous flagellation (such as Pseudomonas, Escherichia, and Faecalibacterium) were extremely sensitive to THPS. The decrease in specific most probable numbers (MPNs) of pathogens (total coliforms and Escherichia coli) with the increase in THPS concentration also proved the sterilization ability of THPS in the sludge pretreatment process. Pretreatment of sludge with concentrations of THPS higher than 37.41 mg/g VSS would meet the pathogen requirements for land application of Class A biosolids.

Co-production of biohydrogen and biomethane from food waste and paper waste via recirculated two-phase anaerobic digestion process: Bioenergy yields and metabolic distribution.

To achieve the co-production of H2 and CH4, co-digestion of food waste (FW) and paper waste (PW) was performed on the recirculated two-phase anaerobic digestion (R-TPAD). The PW content in the feedstock increased from 0% to 20%, 40% and 50% (in total solids) with FW as the rest. The results showed that bioH2 and bioCH4 were simultaneously and stably produced in the long-term operation. With the increasing PW content, the removal efficiency of volatile solids decreased slightly from 84.9% to 78.4%; the bioH2 yields increased from 50 to 79 NL-H2/kg-VSfed while the bioCH4 yields decreased from 426 to 329 NL-CH4/kg-VSfed. With the fixed amount of FW, adding PW could significantly increase the total bioenergy yields. The relative abundance showed that the key H2-producing bacteria, Caproiciproducens and Thermoanaerobacterium, increased after PW addition. The microbial distribution suggests that the H2-producers were recirculated to the first stage after proliferating in the second stage.