Pretreated Sugarcane Straw Research Articles

A Multiomics Perspective on Plant Cell Wall-Degrading Enzyme Production: Insights from the Unexploited Fungus Trichoderma erinaceum.

Trichoderma erinaceum is a filamentous fungus that was isolated from decaying sugarcane straw at a Brazilian ethanol biorefinery. This fungus shows potential as a source of plant cell wall-degrading enzymes (PCWDEs). In this study, we conducted a comprehensive multiomics investigation of T. erinaceum to gain insights into its enzymatic capabilities and genetic makeup. Firstly, we performed genome sequencing and assembly, which resulted in the identification of 10,942 genes in the T. erinaceum genome. We then conducted transcriptomics and secretome analyses to map the gene expression patterns and identify the enzymes produced by T. erinaceum in the presence of different substrates such as glucose, microcrystalline cellulose, pretreated sugarcane straw, and pretreated energy cane bagasse. Our analyses revealed that T. erinaceum highly expresses genes directly related to lignocellulose degradation when grown on pretreated energy cane and sugarcane substrates. Furthermore, our secretome analysis identified 35 carbohydrate-active enzymes, primarily PCWDEs. To further explore the enzymatic capabilities of T. erinaceum, we selected a β-glucosidase from the secretome data for recombinant production in a fungal strain. The recombinant enzyme demonstrated superior performance in degrading cellobiose and laminaribiose compared to a well-known enzyme derived from Trichoderma reesei. Overall, this comprehensive study provides valuable insights into both the genetic patterns of T. erinaceum and its potential for lignocellulose degradation and enzyme production. The obtained genomic data can serve as an important resource for future genetic engineering efforts aimed at optimizing enzyme production from this fungus.

Inhibition of enzyme hydrolysis of cellulose by phenols from hydrothermally pretreated sugarcane straw

Relatively few studies have addressed the characterization of sugarcane straw (SCS) for production of fermentable sugars through enzyme hydrolysis. Straw is a major co-product of the sugarcane harvest in Brazil that has potential to sustainably increase cellulosic feedstocks in Brazil by 50%. Pretreatment of 10% w/v straw with liquid hot water (LHW) at 180 °C for 50 min (severity, So, of 4.05), solubilizes hemicellulose, preserves glucan, and generates 4.49 g/L soluble phenolic compounds in the resulting liquid. Extracts from washing pretreated solids with excess hot water followed by acetone resulted in 1.10 and 0.83 g/L phenolics, respectively. Acetone-derived extracts were more inhibitory and decreased glucose yield for enzyme hydrolysis of Solka Floc (a lignin-free cellulose) by 42%. In comparison, pretreated straw washed with hot water or acetone was readily hydrolyzed to 92% and 97% by cellulase enzyme. Hydrothermally treated SCS has the potential to provide a valuable and added source of fermentable sugars suitable for bioprocessing into biofuels and bioproducts when cellulase enzyme inhibitors are removed after pretreatment.

Rational feeding strategies of substrate and enzymes to enzymatic hydrolysis bioreactors

Bioreactors operating in fed-batch mode improve the enzymatic hydrolysis productivity at high biomass loadings. The present work aimed to apply rational feeding strategies of substrates (pretreated sugarcane straw) and enzymes (CellicCtec2?) to achieve sugar titers at industrial levels. The instantaneous substrate concentration was kept constant at 5% (w/v) along the fed-batch. The enzyme dosage inside the bioreactor was adjusted so that the reaction rate was not less than a pre-defined value (a percentage of the initial reaction rate ? rmin). When r reached values below rmin, enzyme pulses were applied to return the reaction rate to its initial value (r0). The optimized feeding policy indicated a reaction rate maintained at a minimum of 70% of r0, based on the trade-off between glucose productivity and enzyme saving. Initially, it was possible to process a 21% (w/v) solid load, achieving 160 g/L of glucose concentration and 80% of glucose yield. It was verified that non-productive enzyme adsorption was the main reason for some reduction of hydrolysis yield regarding the theoretical cellulose-to-glucose conversion. An increment of 30 g/L in the final glucose concentration was achieved when a lignin-blocking additive (soybean protein) was used in the enzymatic hydrolysis.

Fermentative Production of Lasiodiplodan by Lasiodiplodia theobromae CCT3966 from Pretreated Sugarcane Straw

Lasiodiplodan is a β-glucan polymer with different interesting characteristics, including therapeutic properties. It is an extracellular product, which is produced by the filamentous fungus Lasiodiplodia theobromae, using glucose as a substrate. In the present work, the production of lasiodiplodan was studied by the utilization of sugarcane straw as a low-cost carbon source. Glucose-rich sugarcane straw hydrolysate was obtained by a sequential pretreatment with dilute nitric acid (1% v/v) and sodium hydroxide (1% w/v), followed by enzymatic hydrolysis. The fermentation process was conducted by the cultivation of the strain Lasiodiplodia theobromae CCT3966 in sugarcane straw hydrolysate in a shake flask at 28 °C for 114 h. It was found that hydrolysate obtained after enzymatic hydrolysis contained 47.10 gL−1 of glucose. Fermentation experiments of lasiodiplodan synthesis showed that the peak yield and productivity of 0.054 gg−1 glucose consumed and 0.016 gL−1 h−1, respectively, were obtained at 72 h fermentation time. Fungal growth, glucose consumption, and lasiodiplodan production from sugarcane straw hydrolysate presented a similar pattern to kinetic models. The study on the chemical structure of lasiodiplodan produced showed it had a β-glucan construction. The current study revealed that sugarcane straw is a promising substrate for the production of lasiodiplodan.

Furfural and levoglucosenone production from the pyrolysis of ionic liquid pre-treated sugarcane straw

The current work focuses on the comparison of renewable choline cation-based ionic liquids (ILs) with imidazolium cation-based ILs for their delignification performance. Choline cation-based ILs removed a higher percentage of lignin (78.1–83.7% vs. 51.4–64.5%) and hemicellulose (64.7–72.8% vs. 7.1–29.9%) from sugarcane straw pre-treatment compared to imidazolium cation-based ILs. This was mainly attributed to the presence of fewer alkyl group and extra –OH group in choline cation. In general, ILs pre-treatment significantly increased the volatile matter and reduced fixed carbon in regenerated cellulose-rich material. Higher content of cellulose and higher crystallinity in the case of choline-based ILs enhanced the production of furfural and levoglucosenone from the pyrolysis of regenerated cellulose rich material. On the contrary, the presence of higher content of amorphous cellulose from imidazolium-based ILs, particularly 1-ethyl-3-methylimidazolium acetate produced γ-butyrolactone. Therefore, ILs pre-treatment can be favourable for the selective production of platform chemicals via pyrolysis route.

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

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

Screening of potential endoglucanases, hydrolysis conditions and different sugarcane straws pretreatments for cello-oligosaccharides production

Cello-oligosaccharides (COS) are oligomers with 2 to 6 β-1,4-linked glucose units, with potential applications in the food/feed and bioenergy industrial sectors. In this study, the combination of five heterologous expressed endoglucanases varying the temperature and pH conditions were evaluated by design of experiments for COS production. Afterwards, the best combination was tested to produce COS from different pretreated sugarcane straws: ionic liquid, diluted acid, hydrothermal and steam-explosion. The results showed that steam explosion pretreated sugarcane straw treated with CtCel9R enzyme at 50 °C and pH 5.0 yielded 13.4 mg COS g biomass−1, 5–18-fold higher compared to the other pretreated straws. Under the conditions evaluated, the removal of hemicellulose and decrease in the cellulose crystallinity can benefits the enzymatic hydrolysis. This is the first study that combined the evaluation of different enzymes, conditions, and sugarcane straw pretreatments to optimize COS production in a single step without glucose formation.

Investigation of Rheological Behavior of Untreated and Microwave-Assisted Alkaline Pretreated Sugarcane Straw for Biofuel Production

Biomass slurries, such as those subjected to microwave-assisted alkaline (MAA) pretreatment, will become common substrates for the production of biofuels in the future. While the rheology of acid and ionic liquid pretreated biomass is known, the rheology of alkaline pretreated biomass is not yet reported; hence, the goal of this study was to establish the rheological characteristics of untreated and MAA pretreated sugarcane straw (SC). Using rotational rheometry and rheological models, the rheology of SC slurries was assessed as a function of particle size and insoluble solids concentration. In the range of 5–17% insoluble solids concentration, the slurries were consistently pseudo-plastic (n = 0.33 ± 0.02), possessed yield stress with their flow accurately described by the Casson rheological model (R2 = 0.98–0.99). The apparent viscosity and yield stress increased by two orders of magnitude with an increase in insoluble solids concentration. Essentially, MAA pretreated slurries exhibited significantly higher values of apparent viscosity and yield stress than the untreated ones, in the range of 55–80% and 21–63% for particle sizes of < 63 µm (P63) and 90–180 µm (P90), respectively. Pretreated P63 samples exhibited higher apparent viscosity and yield stress than the P90. On the other hand, for untreated samples, P63 samples had a reduced apparent viscosity than P90 samples. The results of this study definitively reveal that MAA significantly increases the shear rate dependent shear viscosity values along with the yield stress of biomass slurries. This will, therefore, serve as a benchmark for characterizing other MAA pretreated biomass slurries to guide the design of industrial-scale production equipment.

Optimization of microwave-assisted alkali pretreatment followed by acid hydrolysis of sugarcane straw for production of acetone-butanol-ethanol

ABSTRACT The production of acetone-butanol-ethanol (ABE) calls for effective pretreatment and hydrolysis techniques to maximize reducing sugar yields. In this study, optimized microwave-assisted alkali (MAA) pretreatment followed by acid hydrolysis was assessed for the production of reducing sugars from sugarcane straw (SCS). To evaluate effects of MAA pretreatment on SCS composition, response surface methodology (RSM) was employed upon subjecting SCS to 320, 640, and 960 W microwave power, using 1–3% (w/v) NaOH, and residence time of 5, 15, and 25 min. The pretreated SCS was made to undergo acid hydrolysis at 121 °C temperature and reaction time of 10–60 min to release reducing sugars. ABE was produced by anaerobic fermentation of reducing sugars using Clostridium beijerinkii. After pretreatment, maximum responses of 76.3% lignin removal, 21.1% hemicellulose, and 71.9% cellulose were achieved at 640 W microwave power, 2.8% NaOH concentration, and 19 min. The maximum reducing sugar concentration was 46.2 g/L while 18.7 g/L of ABE was produced. The results revealed that optimized MAA pretreatment followed by acid hydrolysis can enhance the yield of reducing sugars for ABE production with no need for costly enzymes.

Ethanol production from sugarcane straw using different configurations of fermentation and techno-economical evaluation of the best schemes

This work analyzed different processes of ethanol fermentation from pretreated sugarcane straw. The conversion process, addressing enzymatic hydrolysis and fermentation of C5 and C6-sugars, and the determination of two economic indicators of the best configuration, were covered in this study. Design of experiments combined with Desirability function were applied to select the best combination of glucose concentration and yield in the enzymatic hydrolysis step, resulting in a glucose yield ∼30g/100 g of initial raw material under conditions of 13% of solid load and 30 FPU/g pretreated SCS. In addition, some fermentation configurations were evaluated. A presaccharification previous to simultaneous saccharification and fermentation (PSF) had a higher result in ethanol concentration and yield. The application of this configuration and fermentation of C5 sugar from the pretreatment step yields 215.05 L/ton SCS. Return of investment and payback period, as economic indicators, suggest that the alternative PSF is suitable for adoption by sugarcane industry under the evaluated conditions.

Biobutanol production from sugarcane straw: Defining optimal biomass loading for improved ABE fermentation

Key objective of this work was to evaluate the use of cellulosic fraction from sugarcane straw pretreated by liquid hot water (LHW) for butanol production via acetone-butanol-ethanol (ABE) fermentation. Separated hydrolysis and fermentation (SHF), and pre-saccharification and simultaneous saccharification and fermentation (PSSF) were investigated at 10 and 15 % w/v biomass loading. For 15 % w/v, the synergistic effect of weak acids and phenolic compounds made the sugarcane straw hydrolysate poorly fermentable. The 10 % w/v solid load was more favorable (∼ 4-fold higher) in both SHF and PSSF strategies with respect to the ABE production, without including a detoxification step. However, PSSF achieved higher ABE titer (10.5 g/L – SHF; 13.5 g/L – PSSF) and productivity (0.09 g/(L.h) – SHF; 0.14 g/(L.h) – PSSF) when compared with SHF. Using best condition (PSSF at 10 % w/v), it would be possible to estimate a yield of 169 L ABE per ton pretreated sugarcane straw (or 84.5 L ABE per ton of raw sugarcane straw), containing 65 L acetone, 95 L butanol, and 9 L ethanol. This result represents a process efficiency of 28 %, based on carbohydrates content in raw material.

Comparative data on effects of alkaline pretreatments and enzymatic hydrolysis on bioemulsifier production from sugarcane straw by Cutaneotrichosporon mucoides

Bioemulsifiers are surface active compounds which could be potentially used in food processing, cosmetic sector and oil recovery. Sugarcane straw (SS), was used as the raw substrate for the production of bio-emulsifiers (BE) by Cutaneotrichosporon mucoides. Three different delignification strategies using dilute sodium hydroxide, sodium sulfite and ammonium hydroxide followed by enzymatic hydrolysis (Cellic CTec 2, 7.5% total solids, 15 FPU/g, 72 h) were studied. Enzyme hydrolysis of ammonium hydroxide pretreated SS showed a maximum of 62.19 ± 0.74 g/l total reducing sugars with 88.35% hydrolytic efficiency (HE) followed by sodium hydroxide (60.06 ± 0.33 g/l; 85.40% HE) and sodium sulfite pretreated SS (57.22 ± 0.52 g/l; 84.71% HE), respectively. The ultrastructure of SS (native and delignified) by fourier transform-infrared and near infrared spectroscopy, revealed notable structural differences. The fermentation of hydrolysates by C. mucoides into bioemulsifiers showing emulsification index (EI) of 54.33%, 48.66% and 32.66% from sodium sulfite, sodium hydroxide, and ammonium hydroxide pretreated SS, respectively.

Experimental optimization and techno-economic analysis of bioethanol production by simultaneous saccharification and fermentation process using sugarcane straw

The present work aims to determine a suitable yield-productivity balance in bioethanol production from hydrothermally pretreated sugarcane straw via pre-saccharification (PS) and simultaneous saccharification and fermentation (SSF). PS experiments were carried out evaluating effects of enzymatic dosage, biomass loading, and PS time. The performance of the whole process (PSSSF) was evaluated based on overall ethanol yield and productivity considering a simultaneous optimization (desirability function) of both variables. The multi-criteria optimization enabled to reach 5.7% w/w ethanol concentration yielding 290 L of ethanol per ton of pretreated sugarcane straw within 45 h of total processing time. Furthermore, a techno-economic analysis was performed under optimized conditions (14.5 FPU/gcellulose, 19.3% w/v biomass loading and 33 h PS time). This process was integrated into a first-generation plant. Although the economic evaluation exhibited a negative performance, a sensitivity analysis indicated that a decrease of 23.3% in operational expenditure would be enough to achieve feasibility.

Lignocellulolytic characterization and comparative secretome analysis of a Trichoderma erinaceum strain isolated from decaying sugarcane straw

The fungus Trichoderma reesei is employed in the production of most enzyme cocktails used by the lignocellulosic biofuels industry today. Despite significant improvements, the cost of the required enzyme preparations remains high, representing a major obstacle for the industrial production of these alternative fuels. In this study, a new Trichoderma erinaceum strain was isolated from decaying sugarcane straw. The enzyme cocktail secreted by the new isolate during growth in pretreated sugarcane straw-containing medium presented higher specific activities of β-glucosidase, endoxylanase, β-xylosidase and α-galactosidase than the cocktail of a wild T. reesei strain and yielded more glucose in the hydrolysis of pretreated sugarcane straw. A proteomic analysis of the two strains' secretomes identified a total of 86 proteins, of which 48 were exclusive to T. erinaceum, 35 were exclusive to T. reesei and only 3 were common to both strains. The secretome of T. erinaceum also displayed a higher number of carbohydrate-active enzymes than that of T. reesei (37 and 27 enzymes, respectively). Altogether, these results reveal the significant potential of the T. erinaceum species for the production of lignocellulases, both as a possible source of enzymes for the supplementation of industrial cocktails and as a candidate chassis for enzyme production.

On the performance of continuous stirred tank reactor and plug flow reactor for chemical reactions characterised by non-elementary kinetics

This manuscript deals with the comparison of the performance of a continuous stirred tank reactor (CSTR) versus plug flow reactor (PFR) for non-elementary reactions carried out in homogeneous phase. In particular, we propose the commented analysis of some case of interest in terms of characteristics of kinetic expressions, plots of the rate of reaction versus degree of conversion, CSTR/PFR space–time ratio, comparison between the rate of reaction and its mean value, non-dimensional parameters and operating conditions. To this end, three case-studies have been chosen: (1) chemical reactions that can be described by kinetic laws resembling the ones known as Michaelis–Menten’s; (2) production of hydrogen bromide as example of chain reaction in gas phase; (3) chemical reactions governed by Monod-like kinetic expressions. The three cases have been scrutinised under the hypothesis of an isothermal, homogeneous, single and globally irreversible reaction with negligible density changes of the reacting system. The discussion also allowed to highlight non-trivial aspects intimately related to the formal expression of the kinetic laws under investigation. The application of the approach here developed to two practical case-studies of literature has been proposed, i.e. the enzymatic hydrolysis of pretreated sugarcane straw and the biomass growth fermentation process for the production of hydrogen.

Assessment of different biomass feeding strategies for improving the enzymatic hydrolysis of sugarcane straw

This work describes improvements in the enzymatic hydrolysis of hydrothermally pretreated sugarcane straw. The experiments were performed in batch and fed-batch operation modes (50 mL and 3 L working volumes) with final solid loadings of 10, 20, and 30% (w/v) and enzyme dosage of 10 FPU/gsubstrate. The fed-batch assays showed the most promising results in terms of glucose production and cellulose-to-glucose conversion. In the experiments using a 3 L working volume, analysis of cellulose-to-glucose conversion, power consumption, and apparent viscosity showed that the best assays were those with smoother feed delivery (substrate plus enzyme) throughout the enzymatic hydrolysis. This strategy resulted in 108.8 g/L of glucose and cellulose-to-glucose conversion of 59.7%, corresponding to a final theoretical ethanol content of 55.6 g/L. The power consumption per unit volume and the apparent viscosity were less than 5 kW/m3 and 35 mPa s, respectively, throughout the hydrolysis. Thus, a more concentrated liquor and a less powerful motor to deliver power during the hydrolysis process were obtained in fed-batch strategy reducing the process energy costs.

Ruminal metagenomic libraries as a source of relevant hemicellulolytic enzymes for biofuel production.

SummaryThe success of second‐generation (2G) ethanol technology relies on the efficient transformation of hemicellulose into monosaccharides and, particularly, on the full conversion of xylans into xylose for over 18% of fermentable sugars. We sought new hemicellulases using ruminal liquid, after enrichment of microbes with industrial lignocellulosic substrates and preparation of metagenomic libraries. Among 150 000 fosmid clones tested, we identified 22 clones with endoxylanase activity and 125 with β‐xylosidase activity. These positive clones were sequenced en masse, and the analysis revealed open reading frames with a low degree of similarity with known glycosyl hydrolases families. Among them, we searched for enzymes that were thermostable (activity at > 50°C) and that operate at high rate at pH around 5. Upon a wide series of assays, the clones exhibiting the highest endoxylanase and β‐xylosidase activities were identified. The fosmids were sequenced, and the corresponding genes cloned, expressed and proteins purified. We found that the activity of the most active β‐xylosidase was at least 10‐fold higher than that in commercial enzymatic fungal cocktails. Endoxylanase activity was in the range of fungal enzymes. Fungal enzymatic cocktails supplemented with the bacterial hemicellulases exhibited enhanced release of sugars from pretreated sugar cane straw, a relevant agricultural residue.

Enzymatic Hydrolysis of Pretreated Sugarcane Straw: Kinetic Study and Semi-Mechanistic Modeling.

Although there are already commercial-scale productions of second generation (2G) ethanol, focusing efforts on process optimization can be of key importance to make the production cost-effective in large scale. In this scenario, mathematical models may be useful in design, scale-up, optimization, and control of bioreactors. For this reason, the aim of this work was to study the kinetics of the enzymatic hydrolysis of cellulose from sugarcane straw. Experiments using hydrothermally pretreated sugarcane (HPS) straw (195°C, 10min, 200rpm) with and without alkaline delignification (4% NaOH m/v, 30min, 121°C) were carried out in shake flasks (50°C, pH5.0, 200rpm). Solid load was varied in a range of 0.8 to 10% (m/v), in initial velocity and long-term assays. Enzyme concentration (Cellic®CTec2) was varied from 5 to 80 filter paper unit (FPU) gcellulose (-1). It was possible to fit Michaelis-Menten (MM), modified MM, with and without competitive inhibition by glucose, and Chrastil models. Chrastil model and modified MM with inhibition (both suitable for heterogeneous system, with high resistance to internal diffusion) showed more appropriate than pseudo-homogeneous MM model. The fitted models were able to identify key features of the hydrolysis process and can be very useful within the perspective of bioreactors engineering.

Kinetic modeling for enzymatic hydrolysis of pretreated sugarcane straw

Abstract A semimechanistic multi-reaction kinetic model was developed for modeling enzymatic hydrolysis of pretreated sugarcane straw (SCS) at high-solids concentration. The model, including: enzyme adsorption, xylose formation, end-product inhibition and substrate reactivity, was adjusted from a reference condition (enzyme concentration of 10 FPU/g-cellulose and solid loading of 15% w/v, dry basis). A simplified model was also proposed to overcome high parameter correlation and improve model fidelity. Predictive capacity of both simplified and original model was verified for different enzyme concentration (5–60 FPU/g-cellulose), solids loading (10 and 20 %) and end-product sugar inhibition (30 and 60 g/L glucose, 10 g/L cellobiose and 10 g/L xylose). The glucose concentration was predicted with reasonable accuracy by the simplified model, even under critical conditions of sugar-inhibition. The model is able to describe batch saccharification of pretreated SCS at high-solids concentrations.