Green solvents extraction-based detoxification to enhance the enzymatic hydrolysis of steam-exploded lignocellulosic biomass and recover bioactive compounds.

A novel strategy for pre-treated biomass detoxification combining emerging green solvents and low environmental impact extraction technologies was evaluated. Steam-exploded biomass was subjected to microwave-assisted or orbital shaking extraction using bio-based or eutectic solvents. The extracted biomass was enzymatically hydrolysed. The potential of this detoxification methodology was studied in terms of phenolic inhibitors extraction and sugar production improvement. The effect of adding a post-extraction water washing step before hydrolysis was also evaluated. Excellent results were achieved when steam-exploded biomass was subjected to the microwave-assisted extraction combined with the washing step. The highest sugar production was achieved when ethyl lactate was used as extraction agent (49.80 ± 3.10 g total sugar/L) over the control (30.43 ± 0.34 g total sugar/L). Results suggested that a detoxification step based on green solvents would be a promising option to extract phenolic inhibitors, which can be revalorized as antioxidants, and improve the sugar production from the extracted pre-treated biomass.

In Vitro Assessment of the Prebiotic Potential of Xylooligosaccharides from Barley Straw.

Barley straw was subjected to hydrothermal pretreatment (steam explosion) processing to evaluate its potential as a raw material to produce xylooligosaccharides (XOS) suitable for use as a prebiotic. The steam explosion pretreatment generated a liquid fraction containing solubilised hemicellulose. This fraction was purified using gel permeation chromatography to obtain a fraction rich in XOS DP2-DP6. The sample was characterised through analytical techniques such as HPAEC-PAD, FTIR and MALDI-TOF-MS. The prebiotic activity was evaluated using in vitro fermentation in human faecal cultures through the quantification of short-chain fatty acid (SCFA) and lactate production, the evolution of the pH and the consumption of carbon sources. The total SCFA production at the end of fermentation (30 h) was 90.1 mM. Positive significant differences between the amount of XOS from barley straw and fructooligosaccharides after incubation were observed.

Lignin-enriched residues from bioethanol production: Chemical characterization, isocyanate functionalization and oil structuring properties.

Lignin-enriched waste products from bioethanol production of agriculture residues were tested as structuring agents in castor oil once functionalized with hexamethylene diisocyanate. Cane bagasse, barley and wheat straw were processed through steam explosion, pre-saccharification and simultaneous saccharification and fermentation (PSSF). Alternatively, cane bagasse was submitted to steam explosion and enzymatic hydrolysis (EH). Several Nuclear Magnetic Resonance techniques were used to characterize both residues and NCO-functionalized counterparts. The ß-O-4'/resinol/phenylcoumaran content and hydroxyphenyl/guaiacyl/syringyl distribution depend on biomass source, pretreatment, and enzymatic hydrolysis. Total hydroxyl content (from 1.23 for cane bagasse to 1.85 for wheat straw residues), aromatic/aliphatic hydroxyl ratio (0.78 for cane bagasse and 0.61 and 0.49 for barley and wheat straw residues, respectively) and S/G ratio (ranging from 0.25 to 0.86) influence the NCO-functionalization and oleogel rheological response. Oleogels obtained with barley straw residues exhibited the highest values of the storage modulus; around 2 × 105 Pa and 104 Pa for 25% and 20% contents, respectively. PSSF process showed weaker modification, leading to softer viscoelastic response compared to EH. These oleogels exhibited rheological properties similar to lubricating greases of different NLGI grades. Therefore, we herein show an integrative protocol for the valorization of lignin-enriched residues from bioethanol production as potential thickeners of lubricating greases.

[Mucinous tubular and spindle cell renal cell carcinoma: two novel cases and a literature review.] / Carcinoma mucinoso tubular y de células fusiformes renal: aportación de 2 nuevos casos y revisión de la literatura.

INTRODUCTION: Mucinous tubularand spindle cell carcinomas (MTSC) are a relativelyrare subtype of renal cell carcinoma (RCC) . Thesetumors are composed by tubular and spindle cellareas within a mucinous stroma. They are generallylow-grade nuclei tumors and limited to the kidney, sothey usually have a favorable prognosis. We report twonew cases of MTSC treated at our institution. MATERIALS AND METHODS: We reviewed therenal tumors surgery database of our hospital from2008 to 2019, selecting patients diagnosed with CMTF.We evaluated clinicopathological data and evolution ofthe patients. We also reviewed the published literatureto compare it with our findings. RESULTS: Two patients were included in the study,a 50-year-old male (case 1) and a 55-year-old female(case 2). The diagnosis was made by ultrasound asan incidental fi nding in case 1 and during the studyof fl ank pain in case 2. Treatment was surgical withpartial and radical nephrectomy respectively. After amean follow-up of 70 months, both patients are aliveand disease-free. CONCLUSION: Mucinous tubular and spindle cellcarcinomas (MTSC) is a rare renal tumor, approximatelytwo hundred cases have been published. Thereare questions yet to be answered about their diagnosisand behaviour so it is of utmost importance to reportnew cases in order to increase our knowledge and improvepatient care.

INTRODUCCIÓN: Los carcinomasmucinosos tubulares y de células fusiformes (CMTF)son un subtipo relativamente infrecuente de carcinomarenal (CCR). Están compuestos por célulastubulares y fusiformes con un estroma mucinoso.Generalmente son tumores de bajo grado y estánlimitados al riñón, por lo que habitualmente tienenbuen pronóstico. Reportamos dos nuevos casos deCMTF tratados en nuestra institución. MATERIAL Y MÉTODOS: Se realizó una revisiónde todos los tumores renales intervenidos en nuestrohospital desde el año 2008 has ta el año 2019, seleccionandolos pacientes diagnosticados de CMTF.Se evaluaron las características clinicopatológicasy la evolución de los pacientes. Así mismo, se realizóuna revisión exhaustiva de la literatura publicadapara comparar los hallazgos descritos con los denuestros casos. RESULTADOS: Dos pacientes fueron incluidos enel estudio, un varón de 50 años ( caso 1) y una mujer de55 años ( caso 2). El diagnóstico fue ecográfico, de forma incidental en el caso 1 y a partir del estudio de undolor en flanco, en el caso 2. El tratamiento fue quirúrgicomediante nefrectomía parcial y radical respectivamente.Tras un seguimiento medio de 70 meses ambospacientes están vivos y libres de enfermedad. CONCLUSIÓN: El carcinoma mucinoso tubular yde células fusiformes (CMTF) es un tumor renal infrecuente,habiéndose publicado aproximadamente doscentenares de casos. Aún hay incógnitas que resolversobre su diagnóstico y comportamiento, de ahí la importanciade reportar nuevos casos que nos permitanaumentar su conocimiento y mejorar el manejo denuestros pacientes.

Production of xylooligosaccharides, bioethanol, and lignin from structural components of barley straw pretreated with a steam explosion.

Barley straw (BS) is a potential source to obtain bioethanol and value-added products such as xylooligosaccharides (XOS) and lignin for application in diverse industries. In this study, BS was submitted to steam explosion pretreatment to valorize the main components of this lignocellulose biomass. For hemicellulose fraction valorization, different combinations of endo-ß-(1,4)-D-xylanase enzyme with accessory enzymes (α-L-arabinofuranosidase, feruloy -esterase and acetylxylan-esterase) have been studied to produce XOS with a low degree of polymerization. The application of accessory enzymes combined with endo-ß-(1,4)-D-xylanase enzymes turned out to be the most effective strategy for the formation of XOS. The solid fraction obtained after the pretreatment was submitted to presacharification and simultaneous saccharification and fermentation process for bioethanol production. The resulting lignin-rich residue was characterized. In this integrated process, 13.0 g XOS (DP2-DP6), 12.6 g ethanol and 16.6 g lignin were obtained from 100 g of BS, achieving the goal of valorizing this agricultural residue.

Valorization of Greenhouse Horticulture Waste from a Biorefinery Perspective.

Greenhouse cultivation and harvesting generate considerable amounts of organic waste, including vegetal waste from plants and discarded products. This study evaluated the residues derived from tomato cultivation practices in Almería (Spain) as sugar-rich raw materials for biorefineries. First, lignocellulose-based residues were subjected to an alkali-catalyzed extrusion process in a twin-screw extruder (100 °C and 6-12% (w/w) NaOH) to assess maximum sugar recovery during the subsequent enzymatic hydrolysis step. A high saccharification yield was reached when using an alkali concentration of 12% (w/w), releasing up to 81% of the initial glucan. Second, the discarded tomato residue was crushed and centrifuged to collect both the juice and the pulp fractions. The juice contained 39.4 g of sugars per 100 g of dry culled tomato, while the pulp yielded an extra 9.1 g of sugars per 100 g of dry culled tomato after an enzymatic hydrolysis process. The results presented herein show the potential of using horticulture waste as an attractive sugar source for biorefineries, including lignocellulose-based residues when effective fractionation processes, such as reactive extrusion technology, are available.

Efficient utilization of hydrolysates from steam-exploded gardening residues for lactic acid production by optimization of enzyme addition and pH control.

The expansion of urban green areas has boosted the accumulation of gardening lignocellulosic residues that could be potentially used to produce platform chemicals like lactic acid. However, when using lignocelluloses, pretreatment step, such as steam explosion, is often needed to favour sugar release. Considering that the conversion of glucose from cellulose has been widely addressed, this work is focused on the valorisation of the steam-exploded gardening liquid fraction rich in hemicellulosic sugars. Since oligomeric sugars are usually solubilized during steam explosion, an enzymatic hydrolysis step was required in some cases to increase the monosaccharides content. Although the presence of inhibitors released during pretreatment (e.g. formic acid) hindered hydrolysis yields, the addition of hemicellulases and the enzyme dosage optimization resulted in 85%, 89% and 95% of glucose, xylose and arabinose release from soluble oligomers, respectively. Lactobacillus pentosus CECT4023T was used for lactic acid fermentation of C6 and C5 sugars from the hydrolysate with the highest sugars concentration, that did not require enzymatic hydrolysis. Xylose consumption was hampered due to the inhibitory effect of acids that produced pH drop. Different pH control systems were applied and automatic NaOH addition in bioreactor resulted in 21 g L-1 of lactic acid (95% of the maximum theoretical yield) that implied 44% increase in lactic acid production when compared with flask fermentation. These results provide new insights for the valorisation of emerging lignocellulosic materials like gardening residues into high added-value products.

Integral process assessment of sugarcane agricultural crop residues conversion to ethanol.

This work focuses a whole process assessment on post-harvesting sugarcane residues for 2G ethanol production by different saccharification-fermentation conditions at high solids loading, performed after steam explosion, alkaline and acidic pretreatments. Carbohydrate recoveries and enzymatic digestibility results showed that alkali and steam explosion pretreatments were effective for the biomass assayed. Due to a significant improvement (60%) of the glucose released by combining hemicellulases and cellulases only after the NaOH pretreatment, the most favorable process settled comprised an alkali-based pretreatment followed by a pre-saccharification and simultaneous saccharification and fermentation (PSSF). The produced ethanol reached 4.8% (w/w) as a result of an 80% conversion of the glucose from the pretreated biomass. Finally, an ethanol concentration of 3.2% (w/w) was obtained by means of a steam explosion followed by PSSF, representing a suitable start point to further develop a low environmental impact alternative for ethanol production.

Integrated production of second generation ethanol and lactic acid from steam-exploded elephant grass.

Elephant grass was subjected to steam explosion to enhance cellulose accessibility and convert it into ethanol. After catalyzed pretreatment at 190 °C for 5 min, enzymatic hydrolysis was carried out using high rate of solid loading combined with different enzyme dosages. Assays employing 20% (w/v) solids loading and an enzyme dosage of 20 FPU g-1 substrate led to a yield of 86.02 g glucose released per 100 g potential glucose in the water insoluble solids. This condition was selected to carry out the simultaneous saccharification and fermentation procedure through S. cerevisiae CAT-1, producing 42.25 g L-1 ethanol with a yield of 74.57% regard to the maximum theoretical. The liquor containing C5 and C6-sugars was successfully converted into lactic acid using L. buchneri NRRL B-30929, resulting in 13.35 g L-1 with a yield of 68.21% in relation to the maximum theoretical.

Evaluation of lignins from side-streams generated in an olive tree pruning-based biorefinery: Bioethanol production and alkaline pulping.

In modern lignocellulosic-based biorefineries, carbohydrates can be transformed into biofuels and pulp and paper, whereas lignin is burned to obtain energy. However, a part of lignin could be converted into value-added products including bio-based aromatic chemicals, as well as building blocks for materials. Then, a good knowledge of lignin is necessary to define its valorisation procedure. This study characterized different lignins from side-streams produced from olive tree pruning bioethanol production (lignins collected from steam explosion pretreatment with water or phosphoric acid as catalysts, followed by simultaneous saccharification and fermentation process) and alkaline pulping (lignins recovered from kraft and soda-AQ black liquors). Together with the chemical composition, the structure of lignins was investigated by FTIR, 13C NMR, and 2D NMR. Bioethanol lignins had clearly distinct characteristics compared to pulping lignins; a certain number of side-chain linkages (mostly alkyl-aryl ether and resinol) accompanied with lower phenolic hydroxyls content. Bioethanol lignins also showed a significant amount of carbohydrates, mainly glucose and protein impurities. By contrast, pulping lignins revealed xylose together with a dramatical reduction of side-chains (some resinol linkages survive) and thereby higher phenol content, indicating rather severe lignin degradation during alkaline pulping processes. All lignins showed a predominance of syringyl units.

Ethanol production from pretreated olive tree wood and sunflower stalks by an SSF process.

Olive tree wood and sunflower stalks are agricultural residues largely available at low cost in Mediterranean countries. As renewable lignocellulosic materials, their bioconversion may allow both obtaining a value-added product, for fuel ethanol, and facilitating their elimination. In this work, the ethanol production from olive tree wood and sunflower stalks by a simultaneous saccharification and fermentation (SSF) process is studied. As a pretreatment, steam explosion at different temperatures was applied. The water insoluble fractions of steam-pretreated sunflower stalks and steamed, delignified olive tree wood were used as substrates at 10% w/v concentration for an SSF process by a cellulolytic commercial complex and Saccharomyces cerevisiae. After 72-h fermentation, ethanol concentrations up to 30 g/L were obtained in delignified steam-pretreated olive tree wood at 230 degrees C and 5 min. Sunflower stalks pretretated at 220 degrees C and 5 min gave maximum ethanol concentrations of 21 g/L in SSF experiments.

Effect of inhibitors released during steam-explosion pretreatment of barley straw on enzymatic hydrolysis.

The influence of the liquid fraction (prehydrolysate) generated during steam-explosion pretreatment (210 degrees C, 15 min) of barley straw on the enzymatic hydrolysis was determined. Prehydrolysate was analyzed for degradation compounds and sugars' content and used as a medium for enzymatic hydrolysis tests after pH adjusting to 4.8. Our results show that the presence of the compounds contained in the prehydrolysate strongly affects the hydrolysis step (a 25% decrease in cellulose conversion compared with control). Sugars are shown to be more potent inhibitors of enzymatic hydrolysis than degradation products.

Ethanol production from steam-explosion pretreated wheat straw.

Bioconversion of cereal straw to bioethanol is becoming an attractive alternative to conventional fuel ethanol production from grains. In this work, the best operational conditions for steam-explosion pretreatment of wheat straw for ethanol production by a simultaneous saccharification and fermentation process were studied, using diluted acid [H2SO4 0.9% (w/w)] and water as preimpregnation agents. Acid- or water-impregnated biomass was steam-exploded at different temperatures (160-200 degrees C) and residence times (5, 10, and 20 min). Composition of solid and filtrate obtained after pretreatment, enzymatic digestibility and ethanol production of pretreated wheat straw at different experimental conditions was analyzed. The best pretreatment conditions to obtain high conversion yield to ethanol (approx 80% of theoretical) of cellulose-rich residue after steam-explosion were 190 degrees C and 10 min or 200 degrees C and 5 min, in acid-impregnated straw. However, 180 degrees C for 10 min in acid-impregnated biomass provided the highest ethanol yield referred to raw material (140 L/t wheat straw), and sugars recovery yield in the filtrate (300 g/kg wheat straw).

Inulin-containing biomass for ethanol production: carbohydrate extraction and ethanol fermentation.

The use of stalks instead of tubers as a source of carbohydrates for ethanol production has been investigated. The inulin present in the stalks of Jerusalem artichoke was extracted with water and the effect of solid-liquid ratio, temperature, and acid addition was studied and optimized in order to attain a high-fructose fermentable extract. The maximum extraction efficiency (corresponding to 35 g/L) of soluble sugars was obtained at 1/6 solid-liquid ratio. Fermentations of hydrolyzed extracts by baker's yeast and direct fermentation by an inulinase activity yeast were also performed and the potential to use this feedstock for bioethanol production assessed. The results show that the carbohydrates derived from Jerusalem artichoke stalks can be converted efficiently to ethanol by acidic hydrolysis followed by fermentation with Saccharomyces cerevisiae or by direct fermentation of inulin using Kluyveromyces marxianus strains. In this last case about 30 h to complete fermentation was required in comparison with 8-9 h obtained in experiments with S. cerevisiae growth on acid extracted juices.

Optimization of uncatalyzed steam explosion pretreatment of rapeseed straw for biofuel production.

Rapeseed straw constitutes an agricultural residue with great potential as feedstock for ethanol production. In this work, uncatalyzed steam explosion was carried out as a pretreatment to increase the enzymatic digestibility of rapeseed straw. Experimental statistical design and response surface methodology were used to evaluate the influence of the temperature (185-215°C) and the process time (2.5-7.5min). According to the rotatable central composite design applied, 215°C and 7.5min were confirmed to be the optimal conditions, considering the maximization of enzymatic hydrolysis yield as optimization criterion. These conditions led to a maximum yield of 72.3%, equivalent to 81% of potential glucose in pretreated solid. Different configurations for bioethanol production from steam exploded rapeseed straw were investigated using the pretreated solid obtained under optimal conditions as a substrate. As a relevant result, concentrations of ethanol as high as 43.6g/L (5.5% by volume) were obtained as a consequence of using 20% (w/v) solid loading, equivalent to 12.4g ethanol/100g biomass.

Effect of binary combinations of selected toxic compounds on growth and fermentation of Kluyveromyces marxianus.

The inhibitory effects of various lignocellulose degradation products on glucose fermentation by the thermotolerant yeast Kluyveromyces marxianus were studied in batch cultures. The toxicity of the aromatic alcohol catechol and two aromatic aldehydes (4-hydroxybenzaldehyde and vanillin) was investigated in binary combinations. The aldehyde furfural that usually is present in relatively high concentration in hydrolyzates from pentose degradation was also tested. Experiments were conducted by combining agents at concentrations that individually caused 25% inhibition of growth. Compared to the relative toxicity of the individual compounds, combinations of furfural with catechol and 4-hydroxybenzaldehyde were additive (50% inhibition of growth). The other binary combinations assayed (catechol with 4-hydroxybenzaldehyde, and vanillin with catechol, furfural, or 4-hydroxybenzaldehyde) showed synergistic effect on toxicity and caused a 60-90% decrease in cell mass production. The presence of aldehydes in the fermentation medium strongly inhibited cell growth and ethanol production. Kluyveromyces marxianus reduces aldehydes to their corresponding alcohols to mitigate the toxicity of these compounds. The total reduction of aldehydes was needed to start ethanol production. Vanillin, in binary combination, was dramatically toxic and was the only compound for which inhibition could not be overcome by yeast strain assimilation, causing a 90% reduction in both cell growth and fermentation.

Ethanol production from olive oil extraction residue pretreated with hot water.

The olive pulp fraction contained in the residue generated in olive oil extraction by a two-step centrifugation process can be upgraded by using the cellulose fraction to produce ethanol and recovering high value phenols (tyrosol and hydroxytyrosol). Olive pulp was pretreated in a laboratory scale stirred autoclave at different temperatures (150-250 degrees C). Pretreatment was evaluated regarding cellulose recovery, enzymatic hydrolysis effectiveness, ethanol production by a simultaneous saccharification and fermentation process (SSF), and phenols recovery in the filtrate. The pretreatment of olive pulp using water at temperatures between 200 degrees C and 250 degrees C enhanced enzymatic hydrolysis. Maximum ethanol production (11.9 g/L) was obtained after pretreating pulp at 210 degrees C in a SSF fed-batch procedure. Maximum hydroxytyrosol recovery was obtained in the liquid fraction when pretreated at 230 degrees C.

Hydrothermal pretreatment conditions to enhance ethanol production from poplar biomass.

Pretreatment has been recognized as a key step in enzyme-based conversion processes of lignocellulose biomass to ethanol. The aim of this study is to evaluate two hydrothermal pretreatments (steam explosion and liquid hot water) to enhance ethanol production from poplar (Populus nigra) biomass by a simultaneous saccharification and fermentation (SSF) process. The composition of liquid and solid fractions obtained after pretreatment, enzymatic digestibility, and ethanol production of poplar biomass pretreated at different experimental conditions was analyzed. The best results were obtained in steam explosion pretreatment at 210 C and 4 min, taking into account cellulose recovery above 95%, enzymatic hydrolysis yield of about 60%, SSF yield of 60% of theoretical, and 41% xylose recovery in the liquid fraction. Large particles can be used for poplar biomass in both pretreatments, since no significant effect of particle size on enzymatic hydrolysis and SSF was obtained.

Effect of lignocellulosic degradation compounds from steam explosion pretreatment on ethanol fermentation by thermotolerant yeast Kluyveromyces marxianus.

The filtrate from steam-pretreated poplar was analyzed to identify degradation compounds. The effect of selected compounds on growth and ethanolic fermentation of the thermotolerant yeast strain Kluyveromyces marxianus CECT 10875 was tested. Several fermentations on glucose medium, containing individual inhibitory compounds found in the hydrolysate, were carried out. The degree of inhibition on yeast strain growth and ethanolic fermentation was determined. At concentrations found in the prehy-drolysate, none of the individual compounds significantly affected the fermentation. For all tested compounds, growth was inhibited to a lesser extent than ethanol production. Lower concentrations of catechol (0.96 g/L) and 4-hydroxybenzaldehyde (1.02 g/L) were required to produce the 50% reduction in cell mass in comparison to other tested compounds.

Comparing cell viability and ethanol fermentation of the thermotolerant yeast Kluyveromyces marxianus and Saccharomyces cerevisiae on steam-exploded biomass treated with laccase.

In this study, the thermotolerant yeast Kluyveromyces marxianus CECT 10875 was compared to the industrial strain Saccharomyces cerevisiae Ethanol Red for lignocellulosic ethanol production. For it, whole slurry from steam-exploded wheat straw was used as raw material, and two process configurations, simultaneous saccharification and fermentation (SSF) and presaccharification and simultaneous saccharification and fermentation (PSSF), were evaluated. Compared to S. cerevisiae, which was able to produce ethanol in both process configurations, K. marxianus was inhibited, and neither growth nor ethanol production occurred during the processes. However, laccase treatment of the whole slurry removed specifically lignin phenols from the overall inhibitory compounds present in the slurry and triggered the fermentation by K. marxianus, attaining final ethanol concentrations and yields comparable to those obtained by S. cerevisiae.