Auto-catalyzed hydrothermal pretreatment (A-HTP) and sulfuric-acid-catalyzed hydrothermal pretreatment (SA-HTP) were applied to quinoa stalks in order to reduce their recalcitrance towards enzymatic saccharification. Prior to pretreatment, quinoa stalks were extracted with either water or a 50:50 (v/v) ethanol–water mixture for removing saponins. Extraction with water or aqueous ethanol, respectively, led to removal of 52 and 75% (w/w) of the saponins contained in the raw material. Preliminary extraction of quinoa stalks allowed for a lower overall severity during pretreatment, and it led to an increase of glucan recovery in the pretreated solids (above 90%) compared with that of non-extracted quinoa stalks (73–74%). Furthermore, preliminary extraction resulted in enhanced hydrolysis of hemicelluloses and lower by-product formation during pretreatment. The enhancement of hemicelluloses hydrolysis by pre-extraction was more noticeable for SA-HTP than for A-HTP. As a result of the pretreatment, glucan susceptibility towards enzymatic hydrolysis was remarkably improved, and the overall conversion values were higher for the pre-extracted materials (up to 83%) than for the non-extracted ones (64–69%). Higher overall conversion was achieved for the aqueous ethanol-extracted quinoa stalks (72–83%) than for the water-extracted material (65–74%).
Biodiesel production currently follows a first-generation model using edible oils as raw materials. Such a production model is unsustainable, considering that it is limited by the high cost of edible oils, competes with the food sector, and is linked to deforestation and other environmental threats. Changing the raw material base to non-edible oils provides an opportunity to increase the sustainability of the biodiesel industry and to avoid conflicts with food production. Processing non-edible oilseeds for extracting the oil to be used for producing biodiesel generates large amounts of residues, such as de-oiled cakes, seed husks, and fruit shells and pods as well as plant stems and leaves resulting from pruning and other agronomy practices. Most of those residues are currently disposed of by burning or used in a suboptimal way. Bioconversion following the sugar platform route, anaerobic digestion, or enzyme production provides means for upgrading them to advanced biofuels and high-added value products. Bioconversion of plant biomass, including oilseed residues, requires pretreatment to enhance their susceptibility to enzymes and microorganisms. This review provides an outlook on bioconversion approaches applicable to different residues of oilseed-bearing plant species. Recent reports on the pretreatment of non-edible oilseed residues for enhancing their bioconversion through either the sugar platform route or anaerobic digestion are critically discussed. This review is based on an exhaustive Web of Science search performed in January–May 2023.
Conditioning with reducing agents allows alleviation of inhibition of biocatalytic processes by toxic by-products generated during biomass pretreatment, without necessitating the introduction of a separate process step. In this work, conditioning of steam-pretreated spruce with sodium sulfite made it possible to lower the yeast and enzyme dosages in simultaneous saccharification and fermentation (SSF) to 1 g/L and 5 FPU/g WIS, respectively. Techno-economic evaluation indicates that the cost of sodium sulfite can be offset by benefits resulting from a reduction of either the yeast load by 0.68 g/L or the enzyme load by 1 FPU/g WIS. As those thresholds were surpassed, inclusion of conditioning can be justified. Another potential benefit results from shortening the SSF time, which would allow reducing the bioreactor volume and result in capital savings. Sodium sulfite conditioning emerges as an opportunity to lower the financial uncertainty and compensate the overall investment risk for commercializing a softwood-to-ethanol process. (C) 2015 The Authors. Published by Elsevier Ltd.
Yeast immobilization with low-cost carrier materials is a suitable strategy to optimize the fermentation of lignocellulosic hydrolysates for the production of second-generation (2G) ethanol. It is defined as the physical confinement of intact cells to a certain region of space (the carrier) with the preservation of their biological activity. This technological approach facilitates promising strategies for second-generation bioethanol production due to the enhancement of the fermentation performance that is expected to be achieved. Using immobilized cells, the resistance to inhibitors contained in the hydrolysates and the co-utilization of sugars are improved, along with facilitating separation operations and the reuse of yeast in new production cycles. Until now, the most common immobilization technology used calcium alginate as a yeast carrier but other supports such as biochar or multispecies biofilm membranes have emerged as interesting alternatives. This review compiles updated information about cell carriers and yeast-cell requirements for immobilization, and the benefits and drawbacks of different immobilization systems for second-generation bioethanol production are investigated and compared.
A halotolerant, exopolysaccharide-producing bacterium isolated from the Salar de Uyuni salt flat in Bolivia was identified as Bacillus atrophaeus using next-generation sequencing. Comparisons indicate that the genome most likely (p-value: 0.0024) belongs to a subspecies previously not represented in the database. The growth of the bacterial strain and its ability to produce exopolysaccharides (EPS) in synthetic media with glucose or xylose as carbon sources, and in hydrolysates of quinoa stalks, was investigated. The strain grew well in all synthetic media, but the growth in glucose was better than that in xylose. Sugar consumption was better when initial concentrations were low. The growth was good in enzymatically produced cellulosic hydrolysates but was inhibited in hemicellulosic hydrolysates produced using hydrothermal pretreatment. The EPS yields were up to 0.064 g/g on initial glucose and 0.047 g/g on initial xylose, and was higher in media with relatively low sugar concentrations. The EPS was isolated and purified by a sequential procedure including centrifugation, cold ethanol precipitation, trichloroacetic acid treatment, dialysis, and freeze-drying. Glucose and mannose were the main sugars identified in hydrolyzed EPS. The EPS was characterized by size-exclusion chromatography, Fouriertransform infrared (FTIR) spectroscopy, heteronuclear single-quantum coherence nuclear magnetic resonance (HSQC NMR) spectroscopy, scanning electron microscopy, X-ray diffraction, and thermogravimetric analysis. No major differences were elucidated between EPS resulting from cultivations in glucoseor-xylose-based synthetic media, while some divergences with regard to molecular-weight averages and FTIR and HSQC NMR spectra were detected for EPS from hydrolysate-based media.
The halotolerant bacterial strain BU-4, isolated from a hypersaline environment, was identified as an exopolysaccharide (EPS) producer. Pretreatment liquids of steam-exploded quinoa stalks and enzymatic hydrolysates of Curupaú sawdust were evaluated as carbon sources for EPS production with the BU-4 strain, and the produced EPS was characterized using FTIR, TGA, and SEM. Cultivation was performed at 30◦C for 48 h, and the cells were separated from the culture broth by centrifugation. EPS was isolated from the cell pellets by ethanol precipitation, and purified by trichloroacetic acid treatment, followed by centrifugation, dialysis, and freeze-drying. EPS production from quinoa stalks-and Curupaú sawdust-based substrates was 2.73 and 0.89 g L−1, respectively, while 2.34 g L−1 was produced when cultivation was performed on glucose. FTIR analysis of the EPS revealed signals typical for polysaccharides, as well as ester carbonyl groups and sulfate groups. High thermal stability, water retention capacity and gel-forming ability were inferred from SEM and TGA. The capability of the halotolerant isolate for producing EPS from pretreatment liquids and hydrolysates was demonstrated, and characterization of the EPS revealed their broad application potential. The study shows a way for producing value-added products from waste materials using a bacterium from a unique Bolivian ecosystem.
This study aimed to develop an energy- and resource-efficient process for the coproduction of edible mushroom, fermentable sugar and solid biofuel from wood residues. A promising potential was revealed for wood ear fungus (Auricularia auricular-judae), which yielded about 200 g mushroom per kg dry birch-based substrate, with concomitant degradation of 76.8 and 85.7% of lignin and xylan, respectively, in the substrate. Substrate pasteurisation by hot-air (85–100 °C) was as effective as by energy intensive autoclaving (121 °C), resulting comparable mushroom growth and degradation of lignocellulose. The spent mushroom substrate (SMS) contained 28–33% glucan, which upon analytical enzymatic saccharification released around 46% of the potentially-achievable glucose, corresponding to a 2.3–fold enzymatic digestibility compared with that of the raw substrate. The solid leftover generated after enzymatic hydrolysis revealed high thermal energy value and promising combustion characteristics, showing a plausibility to be recycled as solid fuel for self-supporting energy system and space heating.
Pretreatment with edible white-rot fungi has advantages in low inputs of energy and chemicals for reducing the recalcitrance of woody biomass for bioethanol production while harvesting protein-rich food. The effectiveness of fungal pretreatment may vary with substrate composition. In this study, birch with or without bark and nitrogen additives were experimentally studied for their effects on shiitake production, substrate lignocellulosic degradation and enzymatic convertibility with cellulolytic enzymes. Whey was added as protein nitrogen and led to successful outcomes, while non-protein nitrogen urea and ammonium-nitrate resulted in mortality of fungal mycelia. The mushroom yields of one harvest were generally comparable between the treatments, averaging 651 g fresh weight per kilogram dry substrate, and high enough as to be profitable. Nitrogen loading (0.5-0.8%, dry mass) negatively affected lignin degradation and enzymatic convertibility and prolonged cultivation/pretreatment time. The added bark (0-20%) showed quadratic correlation with degradation of lignin, xylan and glucan as well as enzymatic digestibility of glucan. Nitrogen loading of < 0.6% led to maximal mass degradation of xylan and lignin at bark ratios of 4-9% and 14-19%, respectively, peak saccharification of glucan at 6-12% and the shortest pretreatment time at 8-13% bark. The designed substrates resulted in 19-35% of glucan mass loss after fungal pretreatment, less than half of the previously reported values. Nitrogen and bark additions can regulate lignocellulose degradation and saccharification of birch-based substrates. The designed substrate composition could considerably reduce cellulose consumption during fungal pretreatment, thus improving bioconversion efficiency.
Formulation of substrates based on three hardwood species combined with modulation of nitrogen content by whey addition (0–2%) was investigated in an experiment designed in D-optimal model for their effects on biological preproceesing of lignocellulosic feedstock by shiitake mushroom (Lentinula edodes) cultivation. Nitrogen loading was shown a more significant role than wood species for both mushroom production and lignocellulose degradation. The fastest mycelial colonisation occurred with no nitrogen supplementation, but the highest mushroom yields were achieved when 1% whey was added. Low nitrogen content resulted in increased delignification and minimal glucan consumption. Delignification was correlated with degradation of syringyl lignin unit, as indicated by a significant reduction (41.5%) of the syringyl-to-guaiacyl ratio after cultivation. No significant changes in substrate crystallinity were observed. The formation of furan aldehydes and aliphatic acids was negligible during the pasteurisation and fungal cultivation, while the content of soluble phenolics increased up to seven-fold.
Spent mushroom substrates (SMS) from cultivation of shiitake (Lentinula edodes) on three hardwood species were investigated regarding their potential for cellulose saccharification and for ethanolic fermentation of the produced hydrolysates. High glucan digestibility was achieved during enzymatic saccharification of the SMSs, which was related to the low mass fractions of lignin and xylan, and it was neither affected by the relative content of lignin guaiacyl units nor the substrate crystallinity. The high nitrogen content in SMS hydrolysates, which was a consequence of the fungal pretreatment, was positive for the fermentation, and it ensured ethanol yields corresponding to 84–87% of the theoretical value in fermentations without nutrient supplementation. Phenolic compounds and acetic acid were detected in the SMS hydrolysates, but due to their low concentrations, the inhibitory effect was limited. The solid leftovers resulting from SMS hydrolysis and the fermentation residues were quantified and characterized for further valorisation.
This study aimed at developing an integrated process of production of edible summer oyster mushroom (Pleurotus pulmonarius) and preprocessing of the substrate lignocellulose for producing 2nd-generation biofuels based on softwood. Sawdust-based mushroom substrates of softwood spruce (Picea abies) versus hardwood alder (Alnus glutinosa) as a reference were used for production of summer oyster mushrooms. The substrates had been either hot-air pasteurised or steam sterilised before growing the mushroom. The potential of using spent substrate (SMS) after harvest for biofuel production was evaluated by examining the lignocellulosic composition and enzymatic convertibility. The biological efficiency of the substrates ranged 14.0-33.8% and no significant difference was observed between the treatments. The fruiting bodies had similar total protein concentrations ranging between 26.0 and 28.5% regardless of differences in treatments. The average mass degradation of Klason lignin and acid soluble lignin in the substrates after mushroom production were 35.0 and 22.6%, respectively. Glucan, the major carbohydrate component, was initially present in concentrations ranging from 24 to 29% of total dry matter and with similar concentrations observed in both alder-based and spruce-based substrates. After mushroom production, a significant difference was observed between the substrates with the lowest consumption of glucan, 3.9% of the initial mass, in the spruce-based substrate. The selective degradation ability of P. pulmonarius on the lignin fraction, rather than the cellulose component of softwood, is suggested in the present study. Between 84 and 126 g glucose was yielded per kg of dry SMS, spruce based substrates resulted a higher yield than alder substrate from enzymatic saccharification of the spent substrates. The heat treatment of the mushroom's substrate had in general a minor impact on the mushroom production and fungal pretreatment of the substrates; hot-air pasteurisation is apparently more energy efficient method than steam sterilisation.
Enzymatic hydrolysis of pretreated sugarcane bagasse was performed to investigate the production of ethanol. The sugarcane bagasse was pretreated in a process combining steam explosion and alkaline delignification. The lignin content decreased to 83%. Fed-batch enzymatic hydrolyses was initiated with 8% (w/v) solids loading, and 10 FPU/g cellulose. Then, 1% solids were fed at 12, 24 or 48 h intervals. After 120 h, the hydrolysates were fermented with Saccharomyces cerevisiae UFPEDA 1238, and a fourfold increase in ethanol production was reached when fed-batch hydrolysis with a 12-h addition period was used for the steam pretreated and delignified bagasse.
Brazil, with 185 million tons of solid residues generation per harvest, is the largest producer of sugarcane in the world. The utilization of this biomass ranges from the extraction of sugarcane juice for application in the ethanol and sugar industry to energy generation and bio-based products synthesis. Sugarcane residues are basically composed of hemicellulose, cellulose and lignin chemical structures that are tightly linked to each other and are responsible for the integrity of the vegetal biomass. The aim of the present work is to show the different relations of the biomass contents from different varieties, cultivated places, soils, harvest season, and climate. For the chemical and elemental determination, 60 bagasse samples were characterized. The different bagasse samples did not show significant variability in their lignocellulosic contents. The results showed that the biomass characterization is an important step to obtain process characteristics.
Candida maltosa was cultivated in the liquid phase of residual brewing yeast, a major brewery residue, to produce biomass and biofilm. Using response surface methodology, the effect of two variables at two different levels was investigated. The independent variables were agitation speed (at 100 and 200 rpm), and aeration (at 1 and 3 L min−1). Aeration was identified to be important for the production of both biomass and biofilm, while agitation was the only factor significantly affecting biofilm production. The maximal production of biofilm (2.33 g L−1) was achieved for agitation of 200 rpm and aeration of 1 L min−1, while the maximum for biomass (16.97 g L−1) was reached for 100 rpm agitation and 3 L min−1 air flow. A logistic model applied to predict the growth of C. maltosa in the exponential phase and the biofilm production, showed a high degree of agreement between the prediction and the actual biomass measured experimentally. The produced biofilms were further characterized using Fourier-transform infrared spectroscopy (FTIR), Scanning Electron Microscopy (SEM) and Thermogravimetric Analysis (TGA). FTIR allowed the identification of methyl, carbonyl ester and sulfate groups, and revealed the presence of uronic acid moieties and glycosidic bonds. Water-retention ability up to relatively high temperatures was revealed by TGA, and that makes the produced biofilm suitable for production of hydrogels. SEM also gave indications on the hydrogel-forming potential of the biofilm.
Lignocellulosic feedstocks are an important resource for biorefining of renewables to bio-based fuels, chemicals, and materials. Relevant feedstocks include energy crops, residues from agriculture and forestry, and agro-industrial and forest-industrial residues. The feedstocks differ with respect to their recalcitrance to bioconversion through pretreatment and enzymatic saccharification, which will produce sugars that can be further converted to advanced biofuels and other products through microbial fermentation processes. In analytical enzymatic saccharification, the susceptibility of lignocellulosic samples to pretreatment and enzymatic saccharification is assessed in analytical scale using high-throughput or semi-automated techniques. This type of analysis is particularly relevant for screening of large collections of natural or transgenic varieties of plants that are dedicated to production of biofuels or other bio-based chemicals. In combination with studies of plant physiology and cell wall chemistry, analytical enzymatic saccharification can provide information about the fundamental reasons behind lignocellulose recalcitrance as well as about the potential of collections of plants or different fractions of plants for industrial biorefining. This review is focused on techniques used by researchers for screening the susceptibility of plants to pretreatment and enzymatic saccharification, and advantages and disadvantages that are associated with different approaches.
Jatropha curcas L. is a tropical plant with considerable potential for producing biodiesel and other products of high economic and social interest. During the biodiesel production process from J. curcas different residues, such as shells and husks are generated. In this work, the physical characterization of J. curcas fruits was performed, and the chemical composition of a mixture of shells and husks was determined. The physical characterization revealed that shells and husks account, respectively, for 25.0 and 27.8% of the fruit weight. The compositional analyses of the material showed a quite high content of glucans (32.8% w/w) and xylans (16.4% w/w), which indicates the potential of J. curcas shells and husks for production of ethanol, xylitol and other glucose- and xylose-derived products. Acid hydrolysis was applied to a mixture of shells and husks under different sulphuric acid concentrations (from 0.5 to 4.5%), temperatures (170-220 degrees C) and time (10-20 min), and the hydrolytic conversion of xylan was evaluated. A zone of experimental conditions giving maximal xylan conversion was identified at around 4% H2SO4, 180 degrees C and reaction time below 10 min.
Abstract Jatropha curcas is a promising source of oil for the biodiesel industry, and the shells of its fruits could be considered for ethanol production. In this work, the composition of J. curcas shells is investigated, and the potential of dilute-sulphuric acid pretreatment for improving the enzymatic hydrolysis of cellulose is evaluated. A Box–Behnken experimental design was used for assessing the effect of temperature (110–150 °C), H2SO4 concentration (0.5–2.5%) and pretreatment time (15–45 min) on the formation of sugars during pretreatment and on the enzymatic conversion of cellulose. Cellulose conversions above 80% were achieved both in the separated enzymatic hydrolysis and in the simultaneous saccharification and fermentation of the pretreated materials. Optimal SSF conversions were predicted for pretreatments at low temperature (136 °C) and moderate acid concentrations (1.5%) and reaction time (30 min). The inclusion of an extraction step prior to the pretreatment revealed a further improvement of the enzymatic conversion of cellulose.
Bioethanol is the most commonly used biofuel. It is an alternative to replace fossil fuels in renewable energy; it can be produced from lignocellulosic feedstock using a biotechnological process. Their participation of microorganisms is crucial in the bioconversion process of fermentation for ethanol production and can involve bacteria, fungi, and yeasts. However, when working within bioethanol processes from lignocellulose feedstock, microorganisms face some challenges, such as high temperature, high solids content, and the ability to ferment sugars for high ethanol concentration. Such challenges will depend on operative strategies, such as simultaneous saccharification and fermentation, separate hydrolysis and fermentation, semi-simultaneous saccharification and fermentation, and consolidated bioprocessing; these are the most common configurations. This review presents different trends of the microbial role, biochemical application, and fermentation operative strategies for bioethanol production of the second generation.
In this work, dilute sulphuric acid prehydrolysis of residual empty pods of Moringa oleifera fruits was investigated as pretreatment for enzymatic hydrolysis of cellulose. In experiments performed at 130–190 °C for 10–30 min, corresponding to a severity range between log Ro = 1.9 and log Ro = 4.2, the effect of pretreatment conditions on the recovery of polysaccharides and on the enzymatic convertibility of cellulose was evaluated. Overall cellulose recovery was above 95% in the pretreatments performed at 130 and 160 °C, and between 87 and 90% in the pretreatments at 190 °C, while xylan recovery in the most severe pretreatments was only 24.7–50.2%. The highest sugar concentration in the acid prehydrolysates (15.0 g/L) was obtained in the pretreatment performed at 160 °C and 20 min. The formation of degradation products was low at mild pretreatment conditions, but it increased with the severity. Furfural concentration reached 4.04 g/L at log Ro = 3.1 and decreased again with a further increase of the pretreatment severity. HMF, formic acid and levulinic acid were formed only in the most severe pretreatments. The pretreatment was effective for improving the enzymatic hydrolysis of cellulose, and the highest conversion (84.3%) was achieved in the material pretreated at mid severity (log Ro = 3.1).
Biochemical conversion of wheat straw was investigated using hydrothermal pretreatment, enzymatic saccharification, and microbial fermentation. Pretreatment conditions that were compared included autocatalyzed hydrothermal pretreatment at 160, 175, 190, and 205 °C and sulfuric-acid-catalyzed hydrothermal pretreatment at 160 and 190 °C. The effects of using different pretreatment conditions were investigated with regard to (i) chemical composition and enzymatic digestibility of pretreated solids, (ii) carbohydrate composition of pretreatment liquids, (iii) inhibitory byproducts in pretreatment liquids, (iv) furfural in condensates, and (v) fermentability using yeast. The methods used included two-step analytical acid hydrolysis combined with high-performance anion-exchange chromatography (HPAEC), HPLC, ultra-high performance liquid chromatography-electrospray ionization-triple quadrupole-mass spectrometry (UHPLC-ESI-QqQ-MS), and pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). Lignin recoveries in the range of 108–119% for autocatalyzed hydrothermal pretreatment at 205 °C and sulfuric-acid-catalyzed hydrothermal pretreatment were attributed to pseudolignin formation. Xylose concentration in the pretreatment liquid increased with temperature up to 190 °C and then decreased. Enzymatic digestibility was correlated with the removal of hemicelluloses, which was almost quantitative for the autocatalyzed hydrothermal pretreatment at 205 °C. Except for the pretreatment liquid from the autocatalyzed hydrothermal pretreatment at 205 °C, the inhibitory effects on Saccharomyces cerevisiae yeast were low. The highest combined yield of glucose and xylose was achieved for autocatalyzed hydrothermal pretreatment at 190 °C and the subsequent enzymatic saccharification that resulted in approximately 480 kg/ton (dry weight) raw wheat straw.
Bagasse, a major by-product of sugarcane-processing industries, has potential to play a significant role as feedstock for production of cellulosic ethanol, platform chemicals, and bio-based commodities. Pretreatment is essential for efficient processing of lignocellulosic feedstocks by biochemical conversion. In this work, auto catalyzed (A-HTP) and dilute sulfuric-acid-catalyzed (SA-HTP) hydrothermal pretreatment of sugarcane bagasse was investigated, setting the temperature (175-205 degrees C) and the time (4-51 min) in such a way that the severity factor (SF) was always maintained at one of three predetermined values (2.8, 3.8, and 4.8). The investigation covered the effects of different operational pretreatment conditions on (i) the formation of sugars and water-soluble bioconversion inhibitors, including newly discovered inhibitors such as formaldehyde and pbenzoquinone, in the pretreatment liquid, (ii) the chemical composition and recovery of constituents in the solid phase, as determined using two-step treatment with sulfuric acid, Py-GC/MS, and solid-state NMR, (iii) pseudo lignin formation, (iv) furan aldehydes in condensates from the gas phase, (v) enzymatic digestibility of pretreated solids, (vi) enzyme inhibition by pretreatment liquids, and (vii) fermentability of pretreatment liquids using Saccharomyces cerevisiae yeast. Glucose and xylose were the predominant sugars in pretreatment liquids from SAHTP and A-HTP, respectively. For A-HTP, the enzymatic digestibility of the pretreated solids was proportional to the SF, while for SA-HTP no clear trend was observed. The best enzymatic digestibility (above 80%) was achieved for A-HTP performed at SF 4.8. The highest total yields of glucose and xylose, the predominant sugars, were achieved for A-HTP at SF 3.8 and temperatures of 190 degrees C and 205 degrees C. The fermentability of the pretreatment liquids by Saccharomyces cerevisiae was lower for SA-HTP than for A-HTP. The investigation suggests that hydrothermal pretreatment of sugarcane bagasse can be performed with good results without addition of sulfuric acid, but that the conditions must be just harsh enough to almost quantitatively solubilize the hemicelluloses.
The effects of the redox environment on acidic hydrothermal pretreatment were investigated in experiments with sugarcane bagasse (190 degrees C, 14 min) and Norway spruce (205 degrees C, 5 min). To modulate the redox environment, pretreatment was performed without gas addition, with N-2 , or with O-2. Analyses covered pretreated solids, pretreatment liquids, condensates, enzymatic digestibility, and inhibitory effects of pretreatment liquids on yeast. Addition of gas, especially O-2 , resulted in increased severity, as reflected by up to 18 percent units lower recoveries of pretreated solids, up to 31 percent units lower glucan recoveries, improved hemicellulose removal, formation of pseudo-lignin, improved overall glucan conversion, and increased concentrations of several microbial inhibitors. Some inhibitors, such as formaldehyde and coniferyl aldehyde, did not, however, follow that pattern. TAC (Total Aromatic Content) values reflected inhibitory effects of pretreatment liquids. This study demonstrates how gas addition can be used to modulate the severity of acidic hydrothermal pretreatment.
Biochemical conversion of lignocellulosic feedstocks to advanced biofuels and other commodities through a sugar-platform process involves a pretreatment step enhancing the susceptibility of the cellulose to enzymatic hydrolysis. A side effect of pretreatment is formation of lignocellulose-derived by-products that inhibit microbial and enzymatic biocatalysts. This review provides an overview of the formation of inhibitory by-products from lignocellulosic feedstocks as a consequence of using different pretreatment methods and feedstocks as well as an overview of different strategies used to alleviate problems with inhibitors. As technologies for biorefining of lignocellulose become mature and are transferred from laboratory environments to industrial contexts, the importance of management of inhibition problems is envisaged to increase as issues that become increasingly relevant will include the possibility to use recalcitrant feedstocks, obtaining high product yields and high productivity, minimizing the charges of enzymes and microorganisms, and using high solids loadings to obtain high product titers.
The extraction of bioactive compounds and cellulose saccharification are potential directions for the valorization of spent mushroom substrate (SMS). Therefore, investigating the suitability of different extraction methods for recovering bioactive compounds from SMS and how the extraction affects the enzymatic saccharification is of uppermost relevance. In this work, bioactive compounds were extracted from Pleurotus spp. SMS using four extraction methods. For Soxhlet extraction (SoE), a 40:60 ethanol/water mixture gave the highest extraction efficiency (EE) (69.9–71.1%) among the seven solvent systems assayed. Reflux extraction with 40:60 ethanol/water increased the extraction yield and EE compared to SoE. A shorter reflux time yielded a higher extraction of carbohydrates than SoE, while a longer time was more effective for extracting phenolics. The extracts from 240 min of reflux had comparable antioxidant activity (0.3–0.5 mM GAE) with that achieved for SoE. Ultrasound-assisted extraction (UAE) at 65 °C for 60 min allowed an EE (~82%) higher than that achieved by either reflux for up to 150 min or SoE. Subcritical water extraction (SWE) at 150 °C resulted in the best extraction parameters among all the tested methods. Vanillic acid and chlorogenic acid were the primary phenolic acids identified in the extracts. A good correlation between the concentration of caffeic acid and the antioxidant activity of the extracts was found. Saccharification tests revealed an enhancement of the enzymatic digestibility of SMS cellulose after the extraction of bioactive compounds. The findings of this initial study provide indications on new research directions for maximizing the recovery of bioactive compounds and fermentable sugars from SMS.
The aim of this work was to evaluate waste office paper as raw material for bioethanol productionusing four strains of Saccharomyces cerevisiae and Spathasporapassalidarum HMD 14.2.Waste paper was hydrolyzed with 1-5% V/V sulfuric acid to 2-10% w/Vbiomass load for 60-120 min. The most significant variable for the total reduced sugar (TRS) was the biomass load, followed by the acid concentration. The pretreatment time did not exert any significant effect on TRS. The hydrolysate obtained with 5% V/V sulfuric acid, 10% w/V biomass load and 1 hour, containing8.45 g/L glucose and 9.27 g/L xylose, was chosen for the fermentations. The fermentation with S. passalidarum resulted in higher ethanol formation (3.54 g/L) than the fermentation with S. cerevisiae, which corresponds to a hypothetical yield of 0.708 g/g glucose. This indicates that S. passalidarum produces ethanol not only from glucose, but also from xylose.
Using local residues as raw materials for biorefineries is important for sustainable development. Quinoa stalks can be considered raw materials of choice for local biorefinery initiatives in Bolivia. This investigation aims at proposing a biorefinery process to be applied to quinoa residues using our know-how on lignocellulose bioconversion and the asset of robust microbes isolated from extreme environments. The proposed process consists in treating quinoa stalks in a sequence including extraction of saponins, acid hydrolysis of hemicelluloses and enzymatic saccharification of cellulose for producing hydrolysates to be used in bioconversion processes with different alternative microbes. Saponins are extracted with aqueous ethanol, the saponins-free material is subjected to sulfuricacid-catalyzed hydrothermal pretreatment for separating a stream of hemicellulosic sugars and a cellulignin stream that is then saccharified with commercial cellulases. The extracted saponins can further be processed to value-added products or can be used in the enzymatic saccharification stage for enhancing cellulose conversion. The produced hydrolysates are suitable substrates for producing bacterial biopolymers or ethanol. Residual lignin from the enzymatic saccharification can be upgraded for value-added applications. The results of this investigation show the potential of biorefining of quinoa residues for producing biopolymers using halotolerant bacteria isolated in Bolivian Altiplano.
Bioconversion in biorefineries is a way to valorize residues from agriculture and food processing. Pretreatment is an important step in the bioconversion of lignocellulosic materials, including crop residues. This Special Issue includes nine articles on several pretreatment and bioconversion approaches applied to different agricultural residues and food-processing by-products. The materials addressed in this collection cover straw from wheat, rye, and miscanthus, olive tree pruning residue, almond shells and husks, avocado waste, sweet sorghum bagasse, soybean meal, and residues of non-edible oilseeds.
Biochemical conversion of lignocellulosic feedstocks to advanced biofuels and other bio-based commodities typically includes physical diminution, hydrothermal pretreatment, enzymatic saccharification, and valorization of sugars and hydrolysis lignin. This approach is also known as a sugar-platform process. The goal of the pretreatment is to facilitate the ensuing enzymatic saccharification of cellulose, which is otherwise impractical due to the recalcitrance of lignocellulosic feedstocks. This review focuses on hydrothermal pretreatment in comparison to alternative pretreatment methods, biomass properties and recalcitrance, reaction conditions and chemistry of hydrothermal pretreatment, methodology for characterization of pretreatment processes and pretreated materials, and how pretreatment affects subsequent process steps, such as enzymatic saccharification and microbial fermentation. Biochemical conversion based on hydrothermal pretreatment of lignocellulosic feedstocks has emerged as a technology of high industrial relevance and as an area where advances in modern industrial biotechnology become useful for reducing environmental problems and the dependence on fossil resources.
Jatropha curcas shells were extracted with water in a pilot-scale reactor and then pretreated with dilute sulphuric acid. The pretreatment was initially investigated with a Box-Behnken experimental design in the range of 110-180 degrees C, 0.1-1.5% H2SO4 and 20-60 min, and then with complementary experiments at 190 degrees C. The glucan recovery was above 87% in all the experimental runs. Xylan solubilisation was 13-20% in the milder pretreatments and up to 45% in the most severe runs. Around 70% cellulose enzymatic conversion, evaluated with commercial cellulases during 72-h hydrolysis, was achieved for the pretreatments at 180 degrees C, and a region with maximal conversion was predicted for around 190 degrees C. For confirming that estimation, a 2(2)-experiment augmented by one central point and parallel pretreatments of pre-extracted and non-extracted shells were performed. The highest cellulose conversion, reached at the central point, was 16.5% higher for the pre-extracted and pretreated material than for the directly pretreated one. The low cellulose crystallinity index (0.79) of the pre-extracted and pretreated shells correlated well with their better enzymatic convertibility.
Three enzyme preparations based on the cellulase complex of Penicillium verruculosum and three Trichoderma reesei-based enzyme cocktails were used for evaluating the enzymatic convertibility of cellulose contained in glycerol- and sulfuric acid-pretreated bagasse. The hydrolysis was initially monitored with a micro-scale method using 2 mL of reaction mixture containing 50 g/L of pretreated solids, and at an enzyme load of 10 mg proteinig cellulose. The results were further validated at a higher scale in a setup consisting of 20 mL of reaction mixture with a substrate concentration of 100 g/L. For all the cellulase preparations, and regardless of the experiment scale, glycerol-pretreated bagasse displayed better enzymatic convertibility than acid-pretreated bagasse. It was observed that when the enzyme load is increased from 2 to 10 mg/g, the cellulose conversion is improved but the specific hydrolysis rate is only marginally affected. Although the Trichoderma-based commercial cocktail CC-3 led to higher hydrolysis rates and conversions than all the other enzyme preparations, the Penicillium-based cellulases, especially PV-Xyl PCA and PV-Hist BGL, also showed good potential. PV-Xyl PCA was relatively effective for hydrolysing acid-pretreated bagasse, and PV-Hist BGL displayed reasonable performance in the hydrolysis in absence of exogenous beta-glucosidase.
Spent mushroom substrate (SMS) is the residual biomass generated after harvesting the fruitbodies of edible/medicinal fungi. Disposal of SMS, the main by-product of the mushroom cultivation process, often leads to serious environmental problems and is financially demanding. Efficient recycling and valorization of SMS are crucial for the sustainable development of the mushroom industry in the frame of the circular economy principles. The physical properties and chemical composition of SMS are a solid fundament for developing several applications, and recent literature shows an increasing research interest in exploiting that inherent potential. This review provides a thorough outlook on SMS exploitation possibilities and discusses critically recent findings related to specific applications in plant and mushroom cultivation, animal husbandry, and recovery of enzymes and bioactive compounds.
A combined pretreatment of sugarcane bagasse with glycerol and sulfuric acid was investigated based on a central composite rotatable experimental design. The following factors were varied: temperature (150–199°C), time (0.69–2.3 h), H2SO4 concentration (0.0–1.1%), and glycerol concentration (55.4–79.6%). Xylans and lignin were considerably solubilized during pretreatment. Xylan solubilization, ranging between 6% and 94%, increased significantly with the increase of temperature, time, and H2SO4 concentration and dropped with the increase of glycerol amount. Glycerol restricted the solubilization and full hydrolysis of xylans and the degradation of xylose. Lignin solubilization (20.6–49.4%) increased with the increase of all the experimental factors. Cellulose recovery, which was generally high, increased with the increasing of glycerol concentration and declined at high levels of the other factors. Recoveries above 97% were achieved at low H2SO4 concentration and high glycerol load, whereas the lowest value (83.4%) was achieved in the longest-lasting experiment. The models based on the experimental results predicted the maximal lignin solubilization at 187.7°C, 2.3 h, 79.6% glycerol, and 0.64% H2SO4, whereas the highest yield of enzymatic hydrolysis can be expected at 194.1°C, 1.67 h, 79.6% glycerol, and 1.1% H2SO4. The optimal conditions were confirmed in control experiments. The synergistic effect of sulfuric acid and glycerol on the enzymatic hydrolysis of cellulose was demonstrated.
Cassava stems are an abundant feedstock that is becoming attractive for biochemical conversion to fuels and chemicals. Since cassava stems are rich in both cellulose and starch, carefully designed pretreatment and digestion procedures are required for achieving high glucan recovery. In this study, partially de-starched cassava stems resulting from a water extraction stage were hydrolyzed with amylases, and the resulting starch-depleted material was pretreated with dilute sulfuric acid, and submitted to enzymatic hydrolysis of cellulose. The effects of acid pretreatment on glucan recovery, enzymatic convertibility, and by-product formation were investigated using a Box-Behnken experimental design with temperature (165-195 degrees C), time (5-35 min), and acid concentration (0.2-1.0%) as independent variables. In further experimental series, the time period was extended up to 110 min while maintaining temperature at 195 degrees C and sulfuric acid concentration at 0.6%. Using those conditions, pretreatment for 50 min gave the best results (83.8% enzymatic convertibility of pretreated cellulose, and (similar to)72% overall glucan-to-glucose conversion).
Chemical characterization of cassava stems from different origin revealed that glucans accounted for 54-63% of the dry weight, whereas 35-67% of these glucans consisted of starch. The cassava stems were subjected to a saccharification study including starch hydrolysis, pretreatment with either sulfuric acid or 1-ethyl-3-methylimidazolium acetate ([Emim]OAc), and enzymatic hydrolysis of cellulose. Starch hydrolysis prior to pretreatment decreased sugar degradation, improved enzymatic convertibility of cellulose, and increased overall glucan conversion. Glucan recovery after pretreatment of starch-free cassava stems (SFCS) was around 85%, but below 52% when the stems were pretreated under the same conditions without preparatory starch hydrolysis. The total amount of hydrolyzed glucan after cellulose hydrolysis was two-fold higher for pretreated SFCS than for directly pretreated stems. Pretreatment with [Emim]OAc resulted in 20% higher glucan conversion than pretreatment with acid. Pyrolysis-GC/MS, X-ray diffraction, CP/MAS C-13 NMR and FTIR analyses revealed major differences between H2SO4- and [Emim]OAc-pretreated material.
Wood chips of Norway spruce were pretreated by steam explosion at 195–215 °C after impregnation with either sulfuric acid (SA) or sulfur dioxide (SD). The effects of different pretreatment conditions on formation of microbial inhibitors were investigated, and the inhibitory effects on yeast of pretreatment liquids and of specific inhibitors that were found in the pretreatment liquids were elucidated. Whereas the concentrations of most inhibitors increased with increasing pretreatment temperatures, there were exceptions, such as formaldehyde and p-hydroxybenzaldehyde. The highest concentration of each inhibitor was typically found in SD-pretreated material, but formic acid was an exception. The toxic effects on yeast were studied using concentrations corresponding to loadings of 12 and 20% total solids (TS). Among individual inhibitors that were quantitated in pretreatment liquids, the concentrations of formaldehyde were by far most toxic. There was no or minimal yeast growth in the formaldehyde concentration range (5.8–7.7 mM) corresponding to 12% TS.
The global production of fossil-based plastics has reached critical levels, and their substitution with bio-based polymers is an urgent requirement. Poly(3-hydroxybutyrate) (PHB) is a biopolymer that can be produced via microbial cultivation, but efficient microorganisms and low-cost substrates are required. Halomonas boliviensis LC1, a moderately halophilic bacterium, is an effective PHB producer, and hydrolysates of the residual stalks of quinoa (Chenopodium quinoa Willd.) can be considered a cheap source of sugars for microbial fermentation processes in quinoa-producing countries. In this study, H. boliviensis LC1 was adapted to a cellulosic hydrolysate of quinoa stalks obtained via acid-catalyzed hydrothermal pretreatment and enzymatic saccharification. The adapted strain was cultivated in hydrolysates and synthetic media, each of them with two different initial concentrations of glucose. Cell growth, glucose consumption, and PHB formation during cultivation were assessed. The cultivation results showed an initial lag in microbial growth and glucose consumption in the quinoa hydrolysates compared to cultivation in synthetic medium, but after 33 h, the values were comparable for all media. Cultivation in hydrolysates with an initial glucose concentration of 15 g/L resulted in a higher glucose consumption rate (0.15 g/(L h) vs. 0.14 g/(L h)) and volumetric productivity of PHB (14.02 mg/(L h) vs. 10.89 mg/(L h)) than cultivation in hydrolysates with 20 g/L as the initial glucose concentration. During most of the cultivation time, the PHB yield on initial glucose was higher for cultivation in synthetic medium than in hydrolysates. The produced PHBs were characterized using advanced analytical techniques, such as high-performance size-exclusion chromatography (HPSEC), Fourier transform infrared (FTIR) spectroscopy, 1H nuclear magnetic resonance (NMR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). HPSEC revealed that the molecular weight of PHB produced in the cellulosic hydrolysate was lower than that of PHB produced in synthetic medium. TGA showed higher thermal stability for PHB produced in synthetic medium than for that produced in the hydrolysate. The results of the other characterization techniques displayed comparable features for both PHB samples. The presented results show the feasibility of producing PHB from quinoa stalks with H. boliviensis.
Hydrolysis lignin, i.e., the hydrolysis residue of cellulosic ethanol plants, was extracted with the green solvent γ-valerolactone (GVL). Treatments at 170–210 °C were performed with either non-acidified GVL/water mixtures (NA-GVL) or with mixtures containing sulfuric acid (SA-GVL). SA-GVL treatment at 210 °C resulted in the highest lignin solubilization (64% (w/w) of initial content), and 76% of the solubilized mass was regenerated by water-induced precipitation. Regenerated lignins were characterized through compositional analysis with sulfuric acid, as well as using pyrolysis–gas chromatography/mass spectrometry (Py-GC/MS), high-performance size-exclusion chromatography (HPSEC), solid-state cross-polarization/magic angle spinning 13C nuclear magnetic resonance (CP/MAS 13C NMR) spectroscopy, 1H–13C heteronuclear single-quantum coherence NMR (HSQC NMR), and Fourier-transform infrared (FTIR) spectroscopy. The characterization revealed that the main difference between regenerated lignins was their molecular weight. Molecular weight averages increased with treatment temperature, and they were higher and had broader distribution for SA-GVL lignins than for NA-GVL lignins.
Sugarcane bagasse was pretreated with dilute phosphoric acid or sulfuric acid to facilitate cellulose hydrolysis and lignin extraction. With phosphoric acid, only 8 % of the initial cellulose was lost after delignification, whereas pretreatment with sulfuric acid resulted in the solubilization of 38 % of the initial cellulose. After enzymatic hydrolysis, the process using phosphoric acid produced approximately 35 % more glucose than that using sulfuric acid. In general, the lignins showed 95–97 % purity (total lignin, w/w), an average molar mass of 9500–10,200 g mol−1, a glass transition temperature of 140–160 °C, and a calorific value of 25 MJ kg−1. Phosphoric acid lignin (PAL) was slightly more polar than sulfuric acid lignin (SAL). PAL had 13 % more oxidized units and 20 % more OH groups than SAL. Regardless of the acid used, the lignins shared similar properties, but differed slightly in the characteristics of their functional groups and chemical bonds. These findings show that pretreatment catalyzed with either of the two acids resulted in lignin with sufficiently good characteristics for use in industrial processes.
The enzymatic hydrolysis of cellulose is inhibited by non-productive adsorption of cellulases to lignin, and that is particularly problematic with lignin-rich materials such as softwood. Although conventional surfactants alleviate non-productive adsorption, using biosurfactants in softwood hydrolysis has not been reported. In this study, the effects of four biosurfactants, namely horse-chestnut escin, Pseudomonas aeruginosa rhamnolipid, and saponins from red and white quinoa varieties, on the enzymatic saccharification of steam-pretreated spruce were investigated. The used biosurfactants improved hydrolysis, and the best-performing one was escin, which led to cellulose conversions above 90%, decreased by around two-thirds lignin inhibition of Avicel hydrolysis, and improved hydrolysis of pretreated spruce by 24%. Red quinoa saponins (RQS) addition resulted in cellulose conversions above 80%, which was around 16% higher than without biosurfactants, and it was more effective than adding rhamnolipid or white quinoa saponins. Cellulose conversion improved with the increase in RQS addition up to 6 g/100 g biomass, but no significant changes were observed above that dosage. Although saponins are known to inhibit yeast growth, no inhibition of Saccharomyces cerevisiae fermentation of hydrolysates produced with RQS addition was detected. This study shows the potential of biosurfactants for enhancing the enzymatic hydrolysis of steam-pretreated softwood.
Steam explosion at 180, 190 and 200 °C for 15 min was applied to sugarcane straw in an industrial sugar/ethanol reactor (2.5 m3). The pretreated straw was delignificated by sodium hydroxide and hydrolyzed with cellulases, or submitted directly to enzymatic hydrolysis after the pretreatment. The pretreatments led to remarkable hemicellulose solubilization, with the maximum (92.7%) for pretreatment performed at 200 °C. Alkaline treatment of the pretreated materials led to lignin solubilization of 86.7% at 180 °C, and only to 81.3% in the material pretreated at 200 °C. All pretreatment conditions led to high hydrolysis conversion of cellulose, with the maximum (80.0%) achieved at 200 °C. Delignification increase the enzymatic conversion (from 58.8% in the cellulignin to 85.1% in the delignificated pulp) of the material pretreated at 180 °C, but for the material pretreated at 190 °C, the improvement was less remarkable, while for the pretreated at 200 °C the hydrolysis conversion decreased after the alkaline treatment.
Sugarcane bagasse was pretreated by steam explosion at 190 degrees C for 15 min and the resulting llulignin was subjected to oxygen-assisted alkaline delignification in a 2(3) factorial design with three ntral points to investigate the effect of temperature, time and initial oxygen pressure on pulp yield, sidual lignin content in the pulp and oxygen incorporation into the recovered lignin. Temperature and ygen pressure had the greatest effect on the results, with optimal conditions resulting in a pulp yield similar to 41 % that contained residual lignin (3.7 %) and oxidized lignin with an oxygen content of 25 The procedure described here produces oxidized lignin with chelating properties and a cellulosic pulp th characteristics that favor pulp dissolution and easy bleaching in one additional step.
This work was aimed to evaluate the effect of the removal of hemicellulose and lignin, by hydrothermal pretreatment, carried out at four different temperatures, namely 180, 185, 190 and 195 °C, for 10 min in a 20-L reactor, and alkaline delignification with 1.0 % (w/v) NaOH, at 100 °C for 1 h, on the enzymatic saccharification of sugarcane bagasse cellulose. For the material pretreated under the most severe conditions (1.0 % (w/v) NaOH, 100 °C, 1 h and 195 °C, 10 min), 95.8 % of the hemicellulosic fraction and 80.9 % of lignin were solubilised upon pretreatment and delignification respectively. The enzymatic conversion of the material obtained under those conditions reached 89.2 % of the initial cellulose, whereas it was 69.2 % for the pretreated but non-delignified material and only 6.0 % for raw bagasse. Models describing the effect of hemicellulose and lignin content on the enzymatic hydrolysis were developed. The statistical analysis of the results emphasized the significance of removal of the hemicellulose and lignin for improving the enzymatic hydrolysis of cellulose.
This article aims to offer a detailed review of bacterial nanocellulose (BNC), addressing its growing global relevance and exploring sustainable approaches through the use of agro-industrial residues as viable cultivation alternatives. BNC is a biopolymer produced by different microorganisms, with Komagateibacter xylinum being the most commonly used in this process. Its distinction in relation to vegetable cellulose lies mainly in its nanometric properties, such as water retention capacity, large surface area and structural resistance. The search for alternative sources has been explored for the large-scale production of biopolymers such as polyhydroxybutyrate (PHB) and exopolysaccharides (EPS) from lignocellulosic biomass. The application of different residues from agroindustry, food and forestry as a source of carbon and nutrients in the biosynthesis of BNC has proven to be a promising strategy to make the production process economically viable. A significant advantage of the BNC biosynthesis process is the virtually natural purity of the cellulose produced, eliminating the need for expensive purification steps. There has been a significant increase in the number of patents related to the use of lignocellulosic biomass, filed by academic institutions and private companies in the last five years. In this context, this study condenses the fundamental principles of BNC, offers a trend analysis through bibliometric review and investigates the current panorama in BNC production, as well as its diverse applications in a wide range of sectors, such as medicine (medical devices, tissue engineering), packaging (biodegradable films, coatings), textiles (smart materials, functional fabrics), construction (sustainable materials), electronics (flexible electronic components) and other innovative areas that benefit from the unique properties of bacterial nanocellulose.
The chemical composition of marabou (Dichrostachys cinerea) wood and its treatment with acetic acid were investigated. Two different treatment approaches, direct acetosolv and combined acid prehydrolysis/acetosolv, were evaluated. The effects of acetic acid concentration (50%, 70% and 90%) and temperature (normal boiling temperature and 121°C) on yield of solids, solubilization of lignin and hemicelluloses and recovery of cellulose were evaluated for both treatments. High solubilization of marabou components was observed in the direct acetosolv treatment at 121°C, especially at the highest acetic acid concentration, where around 84.8% of lignin and 78% of hemicelluloses were removed. When the material was subjected to acid prehydrolysis prior to acetosolv treatment, lignin solubilization was improved, especially at low acetic acid concentrations. Above 80% of the solubilized lignin was recovered from the liquors in the direct acetosolv treatment, but the recovery was lower in the combined treatment. Cellulose was well preserved in all the treatment schemes.
Hot-air (75-100 degrees C) pasteurisation (HAP) of birch-wood-based substrate was compared to conventional autoclaving (steam at 121 degrees C) with regard to shiitake growth and yield, chemical composition of heat-pretreated material and spent mushroom substrate (SMS), enzymatic digestibility of glucan in SMS, and theoretical bioethanol yield. Compared to autoclaving, HAP resulted in faster mycelial growth, earlier fructification, and higher or comparable fruit-body yield. The heat pretreatment methods did not differ regarding the fractions of carbohydrate and lignin in pretreated material and SMS, but HAP typically resulted in lower fractions of extractives. Shiitake cultivation, which reduced the mass fraction of lignin to less than half of the initial without having any major impact on the mass fraction of glucan, enhanced enzymatic hydrolysis of glucan about four-fold. The choice of heating method did not affect enzymatic digestibility. Thus, HAP could substitute autoclaving and facilitate combined shiitake mushroom and bioethanol production.