RESUMO
2,5-Diformylfuran, which can be prepared via the oxidation of biobased HMF, has received considerable attention because of its potential applications in producing furan-based chemicals and functional materials, such as biofuels, polymers, fluorescent material, vitrimers, surfactants, antifungal agents and medicines. This work aimed to develop an efficient one-pot process for chemoenzymatic transformation of biobased substrate to 2,5-diformylfuran with deep eutectic solvent (DES) Betaine:Lactic acid ([BA][LA]) catalyst and oxidase biocatalyst in [BA][LA]-H2O. Using waste bread (50 g/L) and D-fructose (18.0 g/L) as feedstocks in [BA][LA]-H2O (15:85, vol/vol), the yields of HMF were 32.8% (15 min) and 91.6% (90 min) at 150 °C, respectively. These prepared HMF could be biologically oxidized to 2,5-diformylfuran by Escherichia coli pRSFDuet-GOase, achieving a productivity of 0.631 g 2,5-diformylfuran/(g fructose) and 0.323 g 2,5-diformylfuran/(g bread) after 6 h under the mild performance condition. This bioresourced intermediate 2,5-diformylfuran was effectively synthesized from biobased feedstock in an environmentally-friendly system.
Assuntos
Frutose , Furaldeído , Furaldeído/química , Catálise , OxirreduçãoRESUMO
Production of bio-based chemicals from renewable bioresource is a key driver for moving towards sustainable industry. Furfurylamine is known as an important furfural-upgrading product in organic synthesis, as well as monolithic synthetic pharmaceuticals, fibers, additives and polymers. In one-pot manner, biomass was tandemly catalyzed to furfurylamine with sulfonated Sn-PL catalyst and recombinant ω-transaminase biocatalyst. Sn-PL (2.4â¯wt%) catalyzed bamboo shoot shell, corncob and rice straw (75.0â¯g/L) to 76.5-113.0â¯mM furfural at 44.7-58.5 % yield in γ-valerolactone-water (2:8, v:v) at 170 â. The obtained biomass slurries containing furfural were biotransformed to furfurylamine at high yield (0.39-0.42â¯g furfurylamine/g xylan in biomass) with ω-transaminase biocatalyst using isopropylamine (3.0â¯mol isopropylamine/mol furfural) as amine donor at 35 â. Such a chemoenzymatic one-pot process combined the advantages of both solid acids and whole-cells catalysts, which provided an efficient and sustainable approach for preparing an important bio-based furan chemical furfurylamine.
Assuntos
Furaldeído , Transaminases , Óxido de Alumínio , Biomassa , Catálise , Furanos , Dióxido de SilícioRESUMO
In this study, tin-loaded sulfonated zeolite (Sn-zeolite) catalyst was synthesized for catalysis of raw corncob (75.0 g/L) to 103.0 mM furfural at 52.3% yield in water (pH 1.0) at 170 °C. This corncob-derived furfural was subsequently biotransformed with recombinant E. coli CG-19 cells coexpressing NADPH-dependent reductase and glucose dehydrogenase at 35 °C by supplementary of glucose (1.5 mol glucose/mol furfural), sodium dodecyl sulfate (0.50 mM) and NADP+ (1.0 µmol NADP+/mmol furfural) in the aqueous catalytic media (pH 7.5). Both sodium dodecyl sulfate (0.50 mM) and Sn4+ (1.0 mM) could promote reductase activity by 1.4-folds. Within 3 h, furfural was wholly catalyzed into furfuryl alcohol. By combining chemical catalysis with Sn-zeolite and biocatalysis with CG-19 cells in one-pot, an effective and sustainable process was established for tandemly catalyzing renewable biomass into furfuryl alcohol under environmentally-friendly way.
Assuntos
Zea mays , Zeolitas , Catálise , Escherichia coli , Furaldeído , Furanos , NADP , Oxirredutases , EstanhoRESUMO
In this work, an effective hybrid strategy was developed for tandem conversion of biomass to furfurylamine with tin-based solid acid Sn-Maifanitum stone and recombinant Escherichia coli whole cells harboring ω-transaminase. 90.3 mM furfural was obtained from corncob (75 g/L) at 170 °C for 0.5 h over Sn-Maifanitum stone catalyst (3.5 wt%) in the aqueous media (pH 1.0), which could be further bioconverted into furfurylamine at 74.0% yield (based on biomass-derived furfural) within 20.5 h. Finally, an efficient recycling and reuse of Sn-Maifanitum stone catalyst and immobilized Escherichia coli AT2018 whole-cell biocatalyst was developed for the synthesis of furfurylamine from biomass in the one-pot reaction system.
Assuntos
Biomassa , Furanos/metabolismo , Biocatálise , Biotransformação , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , TemperaturaRESUMO
Furfurylamine and furfuryl alcohol are known as important furfural-upgrading derivatives in the production of pharmaceuticals, fibers, additives, polymers, etc. In a one-pot manner, the catalysis of biomass into furan-based chemicals was established in a tandem reaction with sulfonated Sn-graphite catalysts and biocatalysts. Using a raw bamboo shoot shell (75.0 g L-1) as the feedstock, a high furfural yield of 41.1% (based on xylan) was obtained using the heterogeneous Sn-graphite catalyst (3.6 wt% dosage) in water (pH 1.0) for 30 min at 180 °C. Under the optimum bioreaction conditions, the biomass-derived furfural could be transformed into furfuryl alcohol (0.310 g furfuryl alcohol per g xylan in biomass) by a reductase biocatalyst or furfurylamine (0.305 g furfurylamine per g xylan in biomass) using an ω-transaminase biocatalyst. Such one-pot chemoenzymatic processes combined the merits of both heterogeneous catalysts and biocatalysts, and sustainable processes were successfully constructed for synthesizing key bio-based furans.
RESUMO
In this study, organic acid could be used as co-catalyst for assisting solid acid SO42-/SnO2-argil to convert hemicellulose-derived D-xylose into furfural. The relationship between pKa of organic acid and turnover frequency (TOF) of co-catalysis with organic acid plus SO42-/SnO2-argil was explored on the conversion of D-xylose to furfural. Oxalic acid (pKaâ¯=â¯1.25) (0.35â¯wt%) was found to be the optimum co-catalyst for assisting SO42-/SnO2-argil (3.6â¯wt%) to synthesize furfural from D-xylose (20â¯g/L) at 180⯰C for 20â¯min, and the furfural yield and TOF could be obtained at 57.07% and 6.26â¯h-1, respectively. Finally, the obtained furfural (107.6â¯mM) could be completely biotransformed to furfuralcohol by recombinant Escherichia coli CCZU-K14 whole-cells at 30⯰C and pH 6.5 in the presence of 1.5â¯mol glucose/mol furfural and 400â¯mM D-xylose. Clearly, this strategy shows high potential application for the effective synthesis of furfuralcohol from biomass-derived D-xylose.
Assuntos
Ácido Oxálico , Xilose , Catálise , Desidratação , Furaldeído , Humanos , EstanhoRESUMO
A newly synthesized solid acid catalyst SO42-/SnO2-diatomite was prepared for synthesizing furfural from corncob in the presence of homogeneous Brönsted acid. The relationship between pKa of Brönsted acid and turnover frequency (TOF) of co-catalysis with Brönsted acid plus SO42-/SnO2-diatomite was explored on the conversion of corncob to furfural. HCl (pKaâ¯=â¯-7.0) (0.5â¯wt%) plus SO42-/SnO2-diatomite (3.6â¯wt%) gave the highest furfural yield (40.1%) with TOF value at 2.98â¯h-1 in the aqueous media. In the γ-valerolactone-water (6:4, v:v) biphasic media containing 15â¯g/L ZnCl2, one-pot conversion of corncob with co-catalysts gave a furfural yield of 68.9% at 170⯰C for 30â¯min. Additionally, an efficient SO42-/SnO2-diatomite recycling was achieved with a productivity of 15.6â¯gâ¯furfural/(gâ¯solidâ¯acid·day) after 5 cycles of repeated use. Clearly, this one-pot co-catalysis process has high potential application for furfural production in future.
Assuntos
Furaldeído , Estanho , Zea mays , Catálise , Ácido ClorídricoRESUMO
In this study, carbohydrates (cellulose plus hemicellulose) in corncob were effectively converted furfuralcohol (FOL) via chemical-enzymatic catalysis in a one-pot manner. After corncob (2.5 g, dry weight) was pretreated with 0.5 wt% oxalic acid, the obtained corncob-derived xylose (19.8 g/L xylose) could be converted to furfural at 60.1% yield with solid acid catalyst SO42-/SnO2-attapulgite (3.6 wt% catalyst loading) in the water-toluene (3:1, v/v) at 170 °C for 20 min. Moreover, the oxalic acid-pretreated corncob residue (1.152 g, dry weight) was enzymatically hydrolyzed to 0.902 g glucose and 0.202 g arabinose. Using the corncob-derived glucose (1.0 mM glucose/mM furfural) as cosubstrate, the furfural liquor (48.3 mM furfural) was successfully biotransformed to FOL by recombinant Escherichia coli CCZU-A13 cells harboring an NADH-dependent reductase (SsCR) in the water-toluene (4:1, v/v) under the optimum conditions (50 mM PEG-6000, 0.2 mM Zn2+, 0.1 g wet cells/mL, 30 °C, pH 6.5). After the bioreduction for 2 h, FAL was completely converted to FOL. The FOL yield was obtained at 0.11 g FOL/g corncob. Clearly, this one-pot synthesis strategy shows high potential application for the effective synthesis of FOL.
Assuntos
Celulose/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Furanos/síntese química , Oxirredutases/química , Polissacarídeos/química , Tolueno/química , Água/química , Zea mays/química , Furanos/químicaRESUMO
Biocatalytic upgrading of bio-based platform chemical 5-hydroxymethylfurfural (5-HMF) to 2,5-bis(hydroxymethyl)furan (BHMF) is currently of great interest due to the product specificity, mild reaction and high efficiency. In this work, 200mM 5-HMF could be effectively biotransformed to BHMF at 90.6% with highly 5-HMF-tolerant recombinant E. coli CCZU-K14 whole cells at pH 6.5 and 30°C under the optimum reaction conditions (cosubstrate glucose 1.0mol glucose/(mol 5-HMF), D-xylose 400mM, l-glutamic acid 250mM, Mg2+ 1.5mM, 0.2mol ß-cyclodextrin/(mol 5-HMF), CTAB (cetyltrimethyl ammonium bromide) 12.5mM, and 0.1g wet cells/mL). It was found that E. coli CCZU-K14 was highly tolerant to 5-HMF (up to 400mM). Effective bioreduction of biomass-derived 5-HMF (≤200) to BHMF was successfully demonstrated in this study. In conclusion, this strategy showed high potential application for the synthesis of BHMF.
Assuntos
Escherichia coli , Furaldeído/análogos & derivados , Furanos , BiomassaRESUMO
Based on the Prussian blue spectrophotometric method, one high-throughput screening strategy for screening lignin-degrading microorganisms was built on 24-well plate at room temperature. One high activity of alkali lignin-degrading strain Rhodococcus pyridinivorans CCZU-B16 was isolated from soil. After the optimization of biodegradation, 30.2% of alkali lignin (4 g/L) was degraded under the nitrogen-limited condition (30/1 of C/N ratio; g/g) at 30 °C for 72 h. It was found that syringyl (S) units and guaiacyl (G) in lignin decreased after biodegradation. Moreover, the accumulated lipid in cells had a fatty acid profile rich in C16 and C18 with four major constituent fatty acids including palmitic acid (C16:0; 22.4%), palmitoleic acid (C16:1; 21.1%), stearic acid (C18:0; 16.2%), and oleic acid (C18:1; 23.1%). In conclusion, Rhodococcus pyridinivorans CCZU-B16 showed high potential application in future.
Assuntos
Lignina/metabolismo , Rhodococcus/metabolismo , Microbiologia do Solo , Rhodococcus/isolamento & purificaçãoRESUMO
One-pot synthesis of furfuralcohol from corncob-derived xylose was attempted by the tandem catalysis with solid acid SO42-/SnO2-kaoline and recombination Escherichia coli CCZU-T15 whole-cells in the toluene-water media. Using SO42-/SnO2-kaoline (3.5wt%) as catalyst, the furfural yield of 74.3% was obtained from corncob-derived xylose in the toluene-water (1:2, v:v) containing 10mM OP-10 at 170°C for 30min. After furfural liquor was mixed with corncob-hydrolysate from the enzymatic hydrolysis of oxalic acid-pretreated corncob residue, furfural (50.5mM) could be completely biotransformed to furfuralcohol with Escherichia coli CCZU-T15 whole-cells harboring an NADH-dependent reductase (ClCR) in the toluene-water (1:3, v:v) containing 12.5mM OP-10 and 1.6mM glucose/mM furfural at 30°C and pH 6.5. Furfuralcohol was obtained at 13.0% yield based on starting material corncob (100% furfuralcohol yield for bioreduction of furfural step). Clearly, this one-pot synthesis of furfuralcohol strategy shows high potential application for the effective utilization of corncob.
Assuntos
Caulim , Compostos de Estanho , Xilose , Catálise , Escherichia coli , Furaldeído , Tolueno , Água , Zea maysRESUMO
Sugarcane bagasse (SCB) is an abundant, renewable and inexpensive agricultural byproduct for the production of biofuel and other biobased products. To effectively saccharify SCB with cellulases, combination with dilute alkali salts Na2SO3/Na3PO4 (0.4% Na3PO4, 0.03% Na2SO3) at 7.5% sulfidity and hot water (DASHW) in "one-pot" pretreatment media by autoclaving at 110°C for 40min was attempted to pretreat SCB in this study. Furthermore, FT-IR, XRD and SEM were employed to characterize the changes in the cellulose structural characteristics (porosity, morphology, and crystallinity) of the pretreated Na2SO3/Na3PO4-SCB solid residue, which indicated that combination pretreatment could effectively remove lignin and hemicellulose for enhancing enzymatic saccharification. After 72h, the reducing sugars and glucose from the enzymatic in situ hydrolysis of 50g/L Na2SO3/Na3PO4-SCB in dilute Na2SO3/Na3PO4 (0.27% Na3PO4, 0.02% Na2SO3) media were obtained at 33.8 and 21.8g/L, respectively. Finally, the SCB-hydrolysates containing 20g/L glucose were used for ethanol fermentation in the presence of dilute alkali salts. After 48h, the ethanol yield was 0.42g ethanol/g glucose, which represents 82.1% of the theoretical yield. In conclusion, this study provided an effective pretreatment strategy for enhancing SCB's saccharification, which has potential application of other lignocellulosic materials.
Assuntos
Biotecnologia/métodos , Celulose/química , Celulose/metabolismo , Fosfatos/química , Saccharum/química , Sulfatos/química , Fermentação , Temperatura Alta , Sais/química , ÁguaRESUMO
In this study, dilute alkali salts (0.6% NaClO, 0.067% Na2S) pretreatment at 10% sulfidity under the autoclave system at 120°C for 40min was used for pretreating bamboo shoot shell (BSS). Furthermore, FT-IR, XRD and SEM were employed to characterize the changes in the cellulose structural characteristics (porosity, morphology, and crystallinity) of the pretreated BSS solid residue. After 72h, the reducing sugars and glucose from the enzymatic in situ hydrolysis of 50g/L pretreated BSS in dilute NaClO/Na2S media could be obtained at 31.11 and 20.32g/L, respectively. Finally, the obtained BSS-hydrolysates containing alkalic salt NaClO/Na2S resulted in slightly negative effects on the ethanol production. Glucose in BSS-hydrolysates was fermented from 20.0 to 0.17g/L within 48h, and an ethanol yield of 0.41g/g glucose, which represents 80.1% of the theoretical yield, was obtained. This study provided an effective strategy for potential utilization of BSS.
Assuntos
Hipoclorito de Sódio , Sulfetos , Celulase , Etanol , Fermentação , Hidrólise , Ácido Hipocloroso , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Furfuralcohol (FOL) is an important intermediate for the production of lysine, ascorbic acid, and lubricants. It can be used as a hypergolic fuel in rocketry. In this study, it was attempted to synthesize FOL from xylose by tandem catalysis with solid acid SO42-/SnO2-Montmorillonite and recombination Escherichia coli CCZU-K14 whole cells. Using SO42-/SnO2-Montmorillonite (3.0wt% dosage) as catalyst, a highest furfural yield of 41.9% was achieved from xylose at 170°C for 20min. Furthermore, Escherichia coli CCZU-K14 whole cells were used for bioconverting furfural to FOL. The optimum biocatalytic reaction temperature, reaction pH, cosubstrate concentration, and substrate concentration were 30°C, 6.5, 1.5mol glucose/mol furfural, and 200mM, respectively. Finally, the yield of FOL from 200mM furfural was achieved to 100% by Escherichia coli CCZU-K14 whole cells after 24h. In conclusion, this strategy show high potential application for the effective synthesis of FOL.
Assuntos
Furaldeído , Xilose , Ácidos , Biocombustíveis , Catálise , TemperaturaRESUMO
It was the first report that the concentrated hydrolyzates from the enzymatic hydrolysis of dilute NaOH (3wt%)-soaking rice straw at 30°C was used to form [Bmim]PF6-hydrolyzate (50:50, v/v) media for bioconverting ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] (>99% e.e.) with recombinant E. coli CCZU-A13. Compared with pure glucose, the hydrolyzates could promote both initial reaction rate and the intracellular NADH content. Furthermore, emulsifier OP-10 (20mM) was employed to improve the reductase activity. Moreover, Hp-ß-cyclodextrin (0.01mol Hp-ß-cyclodextrin/mol COBE) was also added into this bioreaction system for enhancing the biosynthesis of (R)-CHBE from COBE by E. coli CCZU-A13 whole-cells. The yield of (R)-CHBE (>99% e.e.) from 800mM COBE was obtained at 100% in the [Bmim]PF6-hydrolyzate (50:50, v/v) media by supplementation of OP-10 (20mM) and Hp-ß-CD (8mM). In conclusion, an effective strategy for the biosynthesis of (R)-CHBE was successfully demonstrated.
Assuntos
Acetoacetatos/metabolismo , Meios de Cultura , Escherichia coli/metabolismo , Hidroxibutiratos/metabolismo , Biotransformação , Líquidos Iônicos/metabolismo , OxirreduçãoRESUMO
In this study, sugarcane bagasse (SB) was pretreated with combination pretreatment (e.g., sequential KOH extraction and ionic liquid soaking, sequential KOH extraction and Fenton soaking, or sequential KOH extraction and glycerol soaking). After the enzymatic hydrolysis of pretreated SBs, it was found that all these three concentrated hydrolyzates could be used for the asymmetric bioreduction of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE]. Compared with glucose, arabinose and cellobiose couldn't promote the initial reaction rate, and xylose could increase the intracellular NADH content. Moreover, it was the first report that hydrolyzates could be used for the effective biosynthesis of (S)-CHBE (â¼500g/L; 98.0% yield) from 3000 COBE by whole cells of Escherichia coli CCZU-K14 in the presence of ß-CD (0.4mol ß-CD/mol COBE), l-glutamine (200mM) and glycine (500mM). In conclusion, it is a new alternative to utilize bioresource for the synthesis of key chiral intermediate (S)-CHBE.
Assuntos
Butiratos/metabolismo , Celulose/química , Escherichia coli/citologia , Escherichia coli/metabolismo , Saccharum/química , Biotransformação/efeitos dos fármacos , Carboidratos/análise , Escherichia coli/efeitos dos fármacos , Glutamina/farmacologia , Glicina/farmacologia , Hidrólise , Solubilidade , Fatores de Tempo , beta-Ciclodextrinas/farmacologiaRESUMO
In this study, an effective pretreatment of dilute NaOH-soaked chestnut shell (CNS) with glycerol-HClO4-water (88.8:1.2:10, w/w/w) media at 130 °C for 30 min was successfully demonstrated. Results revealed that the combination pretreatment removed 66.0 % of lignin and 73.7 % of hemicellulose in untreated CNS. The changes in the structural features (crystallinity, morphology, and porosity) of the solid residue of CNS were characterized with Fourier transform infrared spectroscopy, fluorescent microscope, scanning electron microscopy, and X-ray diffraction. Biotransformation of glycerol-HClO4-water pretreated-NaOH-soaked CNS (50 g/L) with a cocktail of enzymes for 72 h, the reducing sugars and glucose were 39.7 and 33.4 g/L, respectively. Moreover, the recovered hydrolyzates containing 20 g/L glucose had no inhibitory effects on the ethanol-fermenting microorganism, and the ethanol production was 0.45 g/g glucose within 48 h. In conclusion, this combination pretreatment shows promise as pretreatment solvent for wheat straw, although the in-depth exploration of this subject is needed.
Assuntos
Etanol/química , Glucose/química , Glicerol/química , Juglans/química , Percloratos/química , Hidróxido de Sódio/química , Lignina/química , Polissacarídeos/química , Água/químicaRESUMO
In this study, it was the first time to report that the cellulases of Galactomyces sp. CCZU11-1 showed high activity and stability in the culture and reaction media containing IL [Mmim]DMP. Using untreated chestnut shell (CNS) as carbon source in the culture media containing IL [Mmim]DMP (5%, w/v), high activity of FPA (28.6U/mL), xylanase (186.2U/mL), and CMCase (107.3U/mL) were obtained, and 184.9mg/L of total protein was achieved. Furthermore, the changes in the structural features (crystallinity, morphology, and porosity) of the solid residue of CNS utilized with Galactomyces sp. CCZU11-1 were characterized with Fourier transform infrared spectroscopy, scanning electron microscopy, and X-ray diffraction. After was enzymatically hydrolyzed with the prepared crude enzymes in IL diluted to 20% (w/v), a high yield of reducing sugars, 62.1%, was obtained. Significantly, Galactomyces sp. CCZU11-1 showed high potential for the efficient transformation of lignocellulosic materials to glucose in a single-step process.
Assuntos
Celulase/química , Celulases/química , Celulose/química , Eleocharis/química , Saccharomycetales/enzimologia , Meios de Cultura , Ensaios Enzimáticos , Hidrólise , Líquidos Iônicos/química , Difração de Raios XRESUMO
In this study, a pretreatment by combining acidified aqueous ionic liquid 1-butyl-3-methylimidazolium chloride (IL [Bmim]Cl) solution with dilute NaOH extraction was employed to pretreat high crystallinity index (CrI) of corn stover before its enzymatic saccharification. After NaOH extraction, [Bmim]Cl-HCl-water (78.8:1.2:20, w/w/w) media was used for further pretreatment at 130 °C for 30 min. After being enzymatically hydrolyzed for 48 h, corn stover pretreated could be biotransformed into reducing sugars in the yield of 95.1%. Furthermore, SEM, XRD and FTIR analyses of untreated and pretreated corn stovers were examined. It was found that the intact structure was disrupted by combination pretreatment and resulted in a porous and amorphous regenerated cellulosic material that greatly improved enzymatic hydrolysis. Finally, the recovered hydrolyzates obtained from the enzymatic hydrolysis of pretreated corn stovers could be fermented into ethanol efficiently. In conclusion, the combination pretreatment shows high potential application in future.
Assuntos
Metabolismo dos Carboidratos/efeitos dos fármacos , Celulase/farmacologia , Imidazóis/farmacologia , Hidróxido de Sódio/farmacologia , Resíduos/análise , Água/farmacologia , Zea mays/química , Cristalização , Etanol/metabolismo , Fermentação/efeitos dos fármacos , HidróliseRESUMO
In this study, it was the first report that Bacillus sp. CCZU11-1 was used for the biotransformation of 1,3-propanediol cyclic sulfate (1,3-PDS) and its derivatives. The catalytic performance of Bacillus sp. sulfatase in the biotransformation of 1,3-PDS was significantly improved by biocatalyst permeabilization and immobilization. Using cell permeabilization, the hydrolytic activity of the whole-cell biocatalyst was increased by 3.5-fold after 1.5 h of pretreatment with 10 % (v/v) toluene at 30 °C and pH 7.0. Biotransformation of 20 mM 1,3-PDS for 24 h, 1,3-propanediol (1,3-PD) could be obtained in the yield of 97.4 % under the optimized reaction condition. Additionally, the immobilized biocatalysts, permeabilized cells entrapped in calcium alginate, and cross-linked enzyme aggregates were further employed to biotansform 1,3-PDS. Moreover, the total operational time of the immobilized biocatalysts could reach above 240 h with high conversion rate (>90 %).