Hydrolysis Of Lignocellulosic Biomass Research Articles

Enzymatic Hydrolysis Performance of Biomass by the Addition of a Lignin Based Biosurfactant

Hydrolysis of biomass for the production of fermentable sugar can be improved by the addition of surfactants. In pulp and paper mills, lignin, which is a by-product of the pulping process, can be utilized as a fine chemical. In the hydrolysis process, lignin is one of the major inhibitors of the enzymatic breakdown cellulose into sugar monomer. Therefore, the conversion of lignin into a biosurfactant offers the opportunity to solve the waste problem and improve hydrolysis efficiency. In this study, lignin derivatives, a biosurfactant, was applied to enzymatic hydrolysis of various lignocellulosic biomass. This Biosurfactant can be prepared by reacting lignin with a hydrophilic polymer such as polyethylene glycol diglycidylethers (PEDGE). In this study, the effect of biosurfactants on the enzymatic hydrolysis of pretreated sweet sorghum bagasse (SSB), oil palm empty fruit bunch, and sugarcane trash with different lignin contents was investigated. The results show that lignin derivatives improve the enzymatic hydrolysis of the pretreated biomass with low lignin content, however, it has less influence on the enzymatic hydrolysis of other pretreated biomass with lignin content higher than 10% (w/w). The use of biosurfactant on SSB kraft pulp can increase the sugar yield from 45.57% to 81.49%.

Enzymatic hydrolysis of several pretreated lignocellulosic biomasses: Fractal kinetic modelling

Enzymatic hydrolysis of three pre-treated lignocellulosic biomasses –LCB- (wheat straw-WS-, corn stover-CSV- and cardoon stems –CS-) is studied. These biomasses were pre-treated by two methods: diluted sulfuric acid and acid ethanol-water extraction at six severity levels (H values). Pretreated solid fractions were hydrolyzed with commercial enzyme cocktails at standard conditions. A first-order kinetic fractal model was fitted to the experimental results. This model accurately describes the hydrolysis of all biomasses at all pre-treatment conditions studied. The results show that the formal first-order kinetic constant k depends on the biomass nature. The hydrolysis rate increases as the pre-treatment severity does, while the fractal exponent value h decreases. With these pre-treatments, and in terms of k and h, WS is highly reactive and, at medium H with EW pretreatment, highly accessible; CSV has a low reactivity and high accessibility and CS has the lowest reactivity and an increasing accessibility as severity rises.

Optimization of cello-oligosaccharides production by enzymatic hydrolysis of hydrothermally pretreated sugarcane straw using cellulolytic and oxidative enzymes

Enzymatic hydrolysis of lignocellulosic biomass accounts for 20–30% of the total cost of second-generation bioethanol production and many efforts have been made in recent years to overcome the high cost of enzymes. Using cello-oligosaccharides (COS), intermediates in cellulose conversion to glucose, may provide advantages over monomeric glucose fermentation, such as lower risk of growth of process contaminants, shorter fermentation time and limited process inhibition by high concentrations of glucose. In addition, COS are also useful as functional oligosaccharides in the food and feed sectors. This study aimed to optimize COS production for further industrial applications. To the best of our knowledge, this is the first study that has used a design of experiments approach to analyze the synergism between endoglucanases, lytic polysaccharide monooxygenase (LPMO), cellobiose dehydrogenase (CDH) and different additives during the hydrolysis of a pretreated sugarcane straw for COS production. After optimization of enzymatic hydrolysis, a combination of the endoglucanases CaCel and CcCel9m, the LPMO TrCel61A, the CDH NcCDHIIa, with lactose and copper as additives, produced 60.49 mg of COS per g of pretreated sugarcane straw, 1.8–2.7-fold more than the commercial enzyme cocktails Cellic® Ctec2 and Celluclast® 1.5 L. The COS/glucose ratio achieved was 298.31, an increase of 3314 and 2294-fold over the commercial enzymatic cocktails, respectively. These results open a new perspective regarding COS production and its industrial application.

Microbial and enzymatic conversion of levulinic acid, an alternative building block to fermentable sugars from cellulosic biomass.

Levulinic acid (LA) is an important chemical building block listed among the top 12 value-added chemicals by the United States Department of Energy, and can be obtained through the hydrolysis of lignocellulosic biomass. Using the same approach as in the catalytic production of LA from biomass, catalytic methods to upgrade LA to higher value chemicals have been investigated. Since the discovery of the catabolic genes and enzymes in the LA metabolic pathway, bioconversion of LA into useful chemicals has attracted attention, and can potentially broaden the range of biochemical products derived from cellulosic biomass. With a brief introduction to the LA catabolic pathway in Pseudomonas spp., this review summarizes the current studies on the microbial conversion of LA into bioproducts, including the recent developments to achieve higher yields through genetic engineering of Escherichia coli cells. Three different types of reactions during the enzymatic conversion of LA are also discussed. KEY POINTS: • Levulinic acid is an alternative building block to sugars from cellulosic biomass. • Introduction of levulinic acid bioconversion with natural and engineered microbes. • Initial enzymatic conversion of levulinic acid proceeds via three different pathways. • 4-Hydroxyvalerate is one of the target chemicals for levulinic acid bioconversion.

Three-Dimensional Imaging of Plant Cell Wall Deconstruction Using Fluorescence Confocal Microscopy

Lignocellulosic biomass (LB) is recalcitrant to enzymatic hydrolysis due to its compact and complex cell wall structure. To identify the parameters behind LB recalcitrance, experimental data over hydrolysis time must be collected. Here, we describe a novel method to collect time-lapse images during cell wall deconstruction by enzymatic hydrolysis. The protocol includes instructions for sample preparation, layout of a custom designed incubation chamber and instructions for confocal time lapse acquisition. The protocol sets out a detailed plan where cross-sections of untreated and pretreated poplar samples are mounted in a sealed frame containing a buffer and an enzymatic cocktail. The sealed frame is then placed into an incubator to maintain the sample at a constant temperature of 50 °C, which is optimal for enzymatic reaction while avoiding enzymatic cocktail evaporation. Using lignin natural autofluorescence, confocal z-stacks of untreated and pretreated samples were acquired at regular time intervals during enzymatic hydrolysis for 24 h. Acquisition parameters were optimized to compromise between image resolution and reduced photo-bleaching. The acquired image might then be processed by further development of algorithms to extract precise quantitative information on cell wall deconstruction. This protocol is an important first step towards elucidating the underlying parameters of LB recalcitrance by allowing the acquisition of high-quality images of LB hydrolysis for extracting quantitative data on LB deconstruction.

Metabolic engineering considerations for the heterologous expression of xylose-catabolic pathways in Saccharomyces cerevisiae.

Xylose, the second most abundant sugar in lignocellulosic biomass hydrolysates, can be fermented by Saccharomyces cerevisiae expressing one of two heterologous xylose pathways: a xylose oxidoreductase pathway and a xylose isomerase pathway. Depending on the type of the pathway, its optimization strategies and the fermentation efficiencies vary significantly. In the present study, we constructed two isogenic strains expressing either the oxidoreductase pathway (XYL123) or the isomerase pathway (XI-XYL3), and delved into simple and reproducible ways to improve the resulting strains. First, the strains were subjected to the deletion of PHO13, overexpression of TAL1, and adaptive evolution, but those individual approaches were only effective in the XYL123 strain but not in the XI-XYL3 strain. Among other optimization strategies of the XI-XYL3 strain, we found that increasing the copy number of the xylose isomerase gene (xylA) is the most promising but yet preliminary strategy for the improvement. These results suggest that the oxidoreductase pathway might provide a simpler metabolic engineering strategy than the isomerase pathway for the development of efficient xylose-fermenting strains under the conditions tested in the present study.

Improving the fermentable sugar yields of wheat straw by high-temperature pre-hydrolysis with thermophilic enzymes of Malbranchea cinnamomea

BackgroundEnzymatic hydrolysis is a key step in the conversion of lignocellulosic polysaccharides to fermentable sugars for the production of biofuels and high-value chemicals. However, current enzyme preparations from mesophilic fungi are deficient in their thermostability and biomass-hydrolyzing efficiency at high temperatures. Thermophilic fungi represent promising sources of thermostable and highly active enzymes for improving the biomass-to-sugar conversion process. Here we present a comprehensive study on the lignocellulosic biomass-degrading ability and enzyme system of thermophilic fungus Malbranchea cinnamomea N12 and the application of its enzymes in the synergistic hydrolysis of lignocellulosic biomass.ResultsMalbranchea cinnamomea N12 was capable of utilizing untreated wheat straw to produce high levels of xylanases and efficiently degrading lignocellulose under thermophilic conditions. Temporal analysis of the wheat straw-induced secretome revealed that M. cinnamomea N12 successively degraded the lignocellulosic polysaccharides through sequential secretion of enzymes targeting xylan and cellulose. Xylanase-enriched cocktail from M. cinnamomea N12 was more active on native and alkali‑pretreated wheat straw than the commercial xylanases from Trichoderma reesei over temperatures ranging from 40 to 75 °C. Integration of M. cinnamomea N12 enzymes with the commercial cellulase preparation increased the glucose and xylose yields of alkali‑pretreated wheat straw by 32 and 166%, respectively, with pronounced effects at elevated temperature.ConclusionsThis study demonstrated the remarkable xylanase-producing ability and strategy of sequential lignocellulose breakdown of M. cinnamomea N12. A new process for the hydrolysis of lignocellulosic biomass was proposed, comprising thermophilic enzymolysis by enzymes of M. cinnamomea N12 followed with mesophilic enzymolysis by commercial cellulases. Developing M. cinnamomea N12 as platforms for thermophilic enzyme mixture production will provide new perspectives for improved conversion yields for current biomass saccharification schemes.

A lytic polysaccharide monooxygenase from Myceliophthora thermophila C1 and its characterization in cleavage of glycosidic chain of cellulose

Lytic polysaccharide monooxygenase (LPMO), a kind of copper-dependent oxidases, has been widely used for the efficient enzymatic hydrolysis of lignocellulosic biomass. In this study, a gene encoding the LPMO from Myceliophthora thermophila C1 (MtC1LPMO) was expressed in Pichia pastoris GS115. A high secretion level of recombinant MtC1LPMO protein was obtained in the culture medium using its native signal peptide. Consequently, MtC1LPMO with a purity of more than 95 % and yield of 46 mg/L was obtained. The optimal enzyme activity was observed at pH 7.5 and 85 °C, and the enzyme displayed remarkable thermostability at 40–60 °C. MtC1LPMO exhibited a synergistic activity with cellulase in the degradation of microcrystalline cellulose, and the reducing sugar content increased by 29 % compared to cellulase alone. Additionally, the results of scanning electron microscopy revealed that MtC1LPMO would cleave the glycosidic chain, leading to subsequent disruption to the surface of microcrystalline cellulose. Finally, the interaction model of MtC1LPMO and cellohexaose was explored using molecular docking simulations, which showed that the residues H1, H68, Y153, H142, Y67, V143, S146, G149, Q151 and P69 of MtC1LPMO played an important role in interacting with cellohexaose.

Production optimization of yellow laccase from Stropharia sp. ITCC 8422 and enzyme-mediated depolymerization and hydrolysis of lignocellulosic biomass for biorefinery application

Biological pretreatment plays a crucial role in utilization of copiously present lignocellulosic biomass in biorefinery. Thus, yellow laccase–producing strain Stropharia sp. ITCC 8422 was studied for its production, purification and integrated depolymerization and hydrolysis. Yellow laccase production optimization using CCD-RSM increased the activity from 160.9 to 323.2 U/L with 2-fold enhancement. Yellow laccase was partially purified using a single-step aqueous two-phase system (ATPS), and PEG 8000 (20%) and Na2SO4 (30%) was the optimum system with activity recovery of 87.8% at the bottom phase of the PEG-salt system. The molecular weight of ATPS partially purified yellow laccase was approximately 100–135 kDa and exhibited stability towards alkaline pH (5 to 11) and temperature (30 to 50 °C) after 12 h of incubation. UV-visible spectra confirmed the production of yellow laccase whereas FTIR and XRD showed stronger intensity of α-helix as compared with β-sheet structure. The depolymerization ability of yellow laccase was determined using wheat straw (WS), rice straw (RS), sugarcane bagasse (SB) and Luffa cylindrica (LC). It effectively depolymerized lignin after 12 h of incubation and was confirmed by kappa number, Klason lignin FTIR and XRD. Later, integrated depolymerization and hydrolysis of lignocellulosic biomass were performed using three different laccase loads, keeping in-house-produced cellulase-xylanase load constant. The increase in laccase load enhanced sugar yield in all lignocellulosic biomass. Thus, one-step depolymerization and hydrolysis have an enormous value in terms of capital, at large-scale biorefinery concept, and enable its utility as a “green tool” and be a step towards “cleaner ecosystem and future”.

Secretome analysis of thermophilic mould Myceliophthora thermophila cultivated on rice straw and hydrolysis of lignocellulosic biomass for bioethanol production

Myceliophthora thermophila encodes for large number of carbohydrate-active enzymes (CAZymes) involved in lignocellulosic biomass degradation. The mould was grown on rice straw in solid state fermentation at pH 5.0 and 45 °C that produced high levels of cellulolytic and xylanolytic enzymes i.e. 2218.12, 515.23, 478.23, 13.34 U/g DMR for xylanase, CMCase, FPase and β-glucosidase, respectively. The secretome analysis of M. thermophila BJAMDU5 by mass spectroscopy, described 124 different proteins with majority of CAZymes consisting of glycosyl hydrolases (GH), lytic polysaccharide mono-oxygenases (LPMO), carbohydrate esterases (CE) and polysaccharide lyases (PL). Furthermore, the enzyme cocktail of the mould was evaluated for hydrolysis of steam treated rice straw that produced 184.59 mg/g substrate reducing sugars after 24 h, which was used for production of bioethanol by using fast fermenting yeast Saccharomyces cerevisiae resulting in high production of bioethanol.

Transformation of xylan into value-added biocommodities using Thermobacillus composti GH10 xylanase

Enzymatic transformation of xylans into renewable fuels and value-added products is mediated by xylanases. Here we describe the biochemical and X-ray structural characterization of Thermobacillus composti GH10 xylanase (TcXyn10A) at 2.1 Å resolution aiming to unravel details of its recognition of glucurono- and arabinoxylan at a molecular level. TcXyn10A improves the efficiency of pretreated lignocellulosic biomass hydrolysis by a commercial enzyme cocktail causing a 15.35 % increase in xylose release and 4.38 % glucose release after 24 h of reaction. The enzyme releases predominantly xylobiose and xylotriose, as well as MeGlcA3 × 3 (from beechwood glucuronoxylan) and a range of decorated xylooligosaccharides (XOS) from rye arabinoxylan, with Ara2 × 2 being the major product. The enzyme liberates XOS with the yields of 29.09 % for beechwood glucuronoxylan and 16.98 % for rye arabinoxylan. Finally, TcXyn10A has a high thermal stability, halotolerance, and resistance to ethanol, biochemical properties that can be desirable for a number of industrial applications.

Sustainable biofuel from microalgae: Application of lignocellulosic wastes and bio-iron nanoparticle for biodiesel production

Biofuels from microalgae are being considered as an alternate renewable energy to fossil fuels, which could extinct by the year 2050 due to its exhaustive usage. Microalgae are tiny cell factories that accumulate large amount of lipids under nutrients starvation, which are sustainable sources for biofuels production. In this study, hydrolysate of lignocellulosic biomass (LCB) was used as a carbon source to improve the production of microalgal biomass, which was further subjected to nitrogen starvation for the enhanced lipid accumulation. The lipid content of LCB supplement 3% and 2% grown cultures of Dictyococcus sp. VSKA18 and Coelastrella sp. M-60 were 44% and 52%, respectively. Further, recovery of fatty acid methyl esters (FAME) was achieved using two different catalysts such as acid (homogeneous) and iron nanoparticles (heterogeneous) synthesized using Sargassum polycystum. Moreover, the saturated fatty acid (SFA) of Dictyococcus sp. VSKA18 and Coelastrella sp. M-60 supplemented with 3% of LCB hydrolysates along with N starvation had yielded 90% and 28% respectively. Fuel quality parameters were also analyzed and compared with ASTM and European standards. Thus, the synthesized bio-iron nanoparticles mediated FAME conversion would be a suitable choice for improving biodiesel production.

Intracellular Redox Perturbation in Saccharomyces cerevisiae Improved Furfural Tolerance and Enhanced Cellulosic Bioethanol Production.

Furfural is a major toxic byproduct found in the hydrolysate of lignocellulosic biomass, which adversely interferes with the growth and ethanol fermentation of Saccharomyces cerevisiae. The current study was focused on the impact of cofactor availability derived intracellular redox perturbation on furfural tolerance. Here, three strategies were employed in cofactor conversion in S. cerevisiae: (1) heterologous expression of NADH dehydrogenase (NDH) from E. coli which catalyzed the NADH to NAD+ and increased the cellular sensitivity to furfural, (2) overexpression of GLR1, OYE2, ZWF1, and IDP1 genes responsible for the interconversion of NADPH and NADP+, which enhanced the furfural tolerance, (3) expression of NAD(P)+ transhydrogenase (PNTB) and NAD+ kinase (POS5) which showed a little impact on furfural tolerance. Besides, a substantial redistribution of metabolic fluxes was also observed with the expression of cofactor-related genes. These results indicated that NADPH-based intracellular redox perturbation plays a key role in furfural tolerance, which suggested single-gene manipulation as an effective strategy for enhancing tolerance and subsequently achieving higher ethanol titer using lignocellulosic hydrolysate.

Novel strategies for glucose production from biomass using heteropoly acid catalyst

Abstract Bioethanol and direct glucose fuel cells pledged clean energy to the world. Cellulose depolymerization for glucose production has been a successful approach in bioethanol production. Heteropoly acids (HPAs) are strong Bronsted solid acid catalysts for biomass hydrolysis. Keggin type HPAs, namely, Silicotungstic acid (HSiW), Phosphotungstic acid (HPW), and Phosphomolybdic acid (HPMo), were used for the hydrolysis of lignocellulosic biomass to glucose. Five different biomass feedstocks, namely, miscanthus, sugarcane leaves, switchgrass, sunflower seeds, and bamboo leaves, were examined for the feasibility of total reducing sugar (TRS) yield through the composition analysis and catalytic biomass hydrolysis. Sunflower seeds contained the maximum holocellulose with 90.6%, and switchgrass contained the least i.e., 77.63%. Among the five biomass tested, switchgrass resulted in the highest TRS (5.77 wt/dry wt. %) with HPMo catalyst at a catalyst to biomass ratio of 30:100 (wt./wt. %), a reaction temperature of 120 °C for 3 h. The reaction parameters for depolymerization were optimized for all three HPAs, and the optimized conditions were 3 h and 120 °C. HPMo showed maximum TRS yield (5.77 wt/dry wt.%) among the three HPAs at 30:100 catalyst to biomass ratio. However, a catalyst to biomass ratio of 20:100 (wt./wt.%) was economical (5.25 wt/dry wt.%) for commercial application.

Evaluation Solvent Level Effect on Sugar Yield during Microwave-Assisted Pretreatment

An efficient pretreatment step is essential to ensure maximum and economical recovery of the fermentable sugar from enzymatic hydrolysis of lignocellulosic biomass. Microwave-assisted pretreatment was found to be a good alternative of conventional heating pretreatment of acid or alkali due to its direct heating of sample which reduce the energy consumption. The present study aims to evaluate the effect of using very diluted solvent (acid and alkali) on monomeric sugar yield during microwave-assisted pretreatment of lignocellulosic biomass (sago palm bark wastes). The changes of the surface structure of untreated and pretreated lignocellulosic biomass were detected using SEM analysis. Three solvents were employed at three concentrations (0.01, 0.05 and 0.1 N). The solvents that utilized in the microwave-assisted pretreatment were sulphuric acid (MA), sodium hydroxide (MH) and sodium bicarbonate (MB) and compared with distilled water (MW). Sago palm bark (SPB) was soaked in these solvents as a substrate at 5% w/v and subjected to microwave power at 800 W in a modified household microwave oven for 5 minutes. Immersing feedstock in distilled water and heating using microwave heating released 5.32 mg/g of monomeric sugar. Highest yield of monomeric sugar was found using 0.1N H2SO4 which yielded 10.28 mg/g during. Applying the pretreatment using the similar concentrations of NaOH and NaHCO3 released 9.35 and 4.31 mg/g of monomeric sugar, respectively. Decreasing chemical solvent level from 0.1 to 0.05 and 0.01 N shows a negative influence on sugar yield for MA and MH pretreatment while MB pretreatment displays a positive effect by extracting some useful components such as xylose.

Modeling of Pretreatment and Acid/Alkaline Hydrolyses of Lignocellulosic Biomasses in Twin-Screw Extruders

A mathematical model for pretreatment and hydrolysis of lignocellulosic biomasses in twin-screw extruders is presented. The model allows for calculation of the concentrations of extracted hemicellu...

Techno‐Economic Analysis for Production of L‐Arabitol from L‐Arabinose

AbstractThe process design for synthesis of arabitol by hydrogenation of arabinose on a supported ruthenium catalyst is described. Aspen HYSYS software was used to design an arabitol production plant for subsequent generation of hydrogen through aqueous phase reforming (APR). The process design included hydrogen recycling requiring a flash drum and recompression. The total costs of arabitol were estimated to be substantially dependent on the feedstock costs and are closely related to arabinose feed cost. Feasible production of hydrogen using APR requires efficient extraction of hemicellulose from lignocellulosic biomass and subsequent hydrolysis.

Biodegradation of recalcitrant compounds and phthalates by culturable bacteria isolated from Liometopum apiculatum microbiota.

Liometopum apiculatum is a species of ants widely distributed in arid and semi-arid ecosystems where there is a relative food shortage compared with tropical ecosystems. L. apiculatum has established an ecological balance involving symbiotic interactions, which have allowed them to survive through mechanisms that are still unknown. Therefore, the aim of this study was to explore the metabolic potential of isolated bacteria from L. apiculatum using enzymatic activity assay and substrate assimilation. Results revealed a complex bacteria consortium belonging to Proteobacteria, Firmicutes, and Actinobacteria phylum. Most of the isolated bacteria showed activities associated with biopolymers degradation, from them Exiguobacterium and B. simplex showed the highest amylolytic activity (27 U/mg protein), while A. johnsonii and B. pumulis showed the highest cellulolytic and xylanolytic activities (1 and 2.9 U/mg protein, respectively). By other hand, some microorganisms such as S. ficaria, E. asburiae, P. agglomerans, A. johnsonii, S. rubidaea, S. marcescens, S. warneri, and M. hydrocarbonoxydans were able to grow up to 1000mg/L of phthalates esters. These results not only revealed the important contribution of the symbionts in L apiculatum ants feeding habits, but also have shown a promising source of enzymes with potential biotechnological applications such as lignocellulosic biomass hydrolysis and bioremediation processes.

Acid Hydrolysis of Lignocellulosic Biomass: Sugars and Furfurals Formation

Hydrolysis of lignocellulosic biomass is a crucial step for the production of sugars and biobased platform chemicals. Pretreatment experiments in a semi-continuous plant with diluted sulphuric acid as catalyst were carried out to measure the time-dependent formation of sugars (glucose, xylose, mannose), furfurals, and organic acids (acetic, formic, and levulinic acid) at different hydrolysis temperatures (180, 200, 220 °C) of one representative of each basic type of lignocellulose: hardwood, softwood, and grass. The addition of the acid catalyst is followed by a sharp increase in the sugar concentration. Xylose and mannose were mainly formed in the initial stages of the process, while glucose was released slowly. Increasing the reaction temperature had a positive effect on the formation of furfurals and organic acids, especially on hydroxymehtylfurfural (HMF) and levulinic acid, regardless of biomass type. In addition, large amounts of formic acid were released during the hydrolysis of miscanthus grass. Structural changes in the solid residue show a complete hydrolysis of hemicellulose at 180 °C and of cellulose at 200 °C after around 120 min reaction time. The results obtained in this study can be used for the optimisation of the hydrolysis conditions and reactor design to maximise the yields of desired products, which might be sugars or furfurals.

Cellulase and oxidative enzymes: new approaches, challenges and perspectives on cellulose degradation for bioethanol production.

Second-generation bioethanol is a sustainable energy source that can be produced from different renewable materials. However, there is a challenge we must overcome to significantly enhance bioethanol production: the hydrolysis of lignocellulosic biomass to fermentable sugars. Synergistic enzymes, such as endoglucanases, β-glucosidases, cellobiohydrolases, and, more recently, lytic polysaccharide monooxygenases and cellobiose dehydrogenases have been used with great success to hydrolyze pretreated biomass. Further advances in the field of second-generation bioethanol production will likely depend on an increased understanding of the interactions between enzymes and lignocellulosic substrates, the development of enzyme engineering, and the optimization of enzyme mixtures to enhance cellulose hydrolysis.