Bioenergy Research Institute

Sugarcane culm has different types of tissue organization, and its heterogeneity can influence the bagasse quality generated in the sugar/ethanol industry. The heterogeneity of the sugarcane bagasse contributes to the intrinsic recalcitrance of the biomass, impairing its conversion to ethanol. To determine the effects of sugarcane culm heterogeneity, the internode and node fractions were pretreated with acid, alkali, and peroxide. The investigation focused on the change in the fraction morphology, crystallinity, chemical composition, and enzymatic digestibility. Scanning electron microscopy (SEM) revealed different anatomical traits between the node and the internode. Vascular bundles appeared in larger number and diameter in the periphery of both the node and the internode. The internode and node responded differently to the pretreatments, with slight differences in the amount of hemicellulose and lignin removal. Furthermore, the internode was more susceptible to the acid and alkaline pretreatments than the node, generating a material with better digestibility. The fractions pretreated with peroxide showed similar enzymatic digestibility. In SEM images, the internode showed more structural damage after pretreat-ments than the node fibers. These results provide insight that pretreated sugarcane bagasse has fractions with different enzymatic digestibility related to their anatomy, and that conversion of the entire biomass is not feasible.

The enzymatic hydrolysis (EH) rate normally decreases during the hydrolysis, leaving unhydrolyzed material as residue. This phenomenon occurs during the hydrolysis of both cellulose (avicel) and lignocellulosic material, in nature or even pretreated. The progression of EH of steam pretreated sugarcane bagasse was associated with an initial (fast), intermediate (slower) and recalcitrant (slowest) phases, at glucan to glucose conversion yields of 61.7, 81.6 and 86%, respectively. Even though the EH of avicel as a simpler material than steam pretreated sugarcane bagasse, EH slowdown was present. The less thermo-stable endo-xylanase lost 58% of initial enzyme activity, followed by b-glucosidase that lost 16%, culminating in FPase activity loss of 30% in the first 24 hours. After 72 hours of EH the total loss of FPase activity was 40% compared to the initial activity. Analysis of the solid residue from EH showed that lignin content, phenolic compounds and ash increased while glucan decreased as hydrolysis progressed. During the initial fast phase of EH, the total solid residue surface area consisted predominantly of internal surface area. Thereafter, in the intermediate and recalcitrant phases of EH, the ratio of external:internal surface area increased. The proposed fiber damage and decrease in internal surface area, probably by EH action, was visualized by scanning electron microscopy imagery. The higher lignin/glucan ratio as EH progressed and enzyme deactivation by thermo instability were the main effects observed, respectively to substrate and enzyme.

The structural and physicochemical characteristics are associated with resistance of plant cell walls to saccharification by enzymes. The effect of physicochemical properties on glucose yield of bagasse from different varieties of sugarcane at low and high enzyme dosages was investigated. The result showed that glucose yield at low enzyme dosage was positively linear correlated with the yield at high enzyme dosage, for both the untreated and pretreated materials. The pretreatment significantly increased the accessibility of substrates by enzyme due to the increase of internal and external surface area. Glucose yield also showed a linear correlation with dye adsorption. However, the increase in glucose yield as a result of pretreatment did not correlate with the increases in crystallinity index and decreases in degree of polymerization. The Principal Component Analysis of infrared data indicated that lignin was the main component that differentiated the varieties before and after pretreatment. These results suggested that the key differences in pretreatment responses among varieties could be mainly attributed to their differences in the internal and external surface area after pretreatment.

Sweet sorghum bagasse (SSB) is considered a potential lignocellulosic raw material for the production of fuel ethanol by means of pretreatment, enzymatic hydrolysis and fermentation. Enzyme cocktail
composition is critical to achieve maximum sugar yield per gram of enzyme used. Given that the enzyme composition relies on feedstock properties and pretreatment applied, the main objective of this study
was to develop an optimised combination of cellulase (Cellic CTec2) and endo-xylanase (CellicHTec2) enzyme preparations (cocktail) that is adapted to steam treated sorghum bagasse. This was done
by using a central composite design by which combined effects of Cellic CTec2 and Cellic HTec2 were investigated further using response surface methodology (RSM). The optimum was defined as the minimum
total enzyme dosage that is needed for a target glucan conversion of at least 80% for an incubation period of 72 h. The addition of Cellic HTec2 proved to boost the glucan conversion to glucose by Cellic CTec2, but synergistic interaction was only observed at early stages of the enzymatic hydrolysis and mainly for those combinations with lower cellulase content. The pretreatment and enzymatic hydrolysis
with 0.15 mL/g WIS (water insoluble solids) of Cellic CTec2 (24.9 mg protein/g WIS) and 0.32 mL/g WIS of Cellic HTec2 (54 mg protein/g WIS), respectively, resulted in 364.8 kg sugar/ton of bagasse, 20% more
than with a conventional enzyme combination.

The extraction of hemicelluloses prior to pulping is an attractive process to be incorporated in the pulp and paper mills to generate valuable by-products such as xylose and xylo-oligomers in addition to core products. This paper investigates the impact of xylan extraction by dilute acid on cooking conditions, pulp yield and properties during kraft and soda-AQ pulping of Eucalyptus grandis wood chips. Modified soda-AQ pulping of the wood chips from which 21.3% of xylan was extracted using a low alkali charge maintained the pulp yield, viscosity and kappa number of non-extracted wood chips. The combination of these processes resulted in chemical reduction while increasing the digester capacity by 12%. Due to extraction, the handsheet properties were reduced, especially the tear index. The degree of hemicellulose pre-extraction and pulping conditions can be optimized based on final paper application.

The structural and physicochemical characteristics are associated with resistance of plant cell walls to saccharification by enzymes. The effect of physicochemical properties on glucose yield of bagasse from different varieties of sugarcane at low and high enzyme dosages was investigated. The result showed that glucose yield at low enzyme dosage was positively linear correlated with the yield at high enzyme dosage, for both the untreated and pretreated materials. The pretreatment significantly increased the accessibility of substrates by enzyme due to the increase of internal and external surface area. Glucose yield also showed a linear correlation with dye adsorption. However, the increase in glucose yield as a result of pretreatment did not correlate with the increases in crystallinity index and decreases in degree of polymerization. The Principal Component Analysis of infrared data indicated that lignin was the main component that differentiated the varieties before and after pretreatment. These results suggested that the key differences in pretreatment responses among varieties could be mainly attributed to their differences in the internal and external surface area after pretreatment.

Spent coffee ground (SCG), a present waste stream from instant coffee production, represents a potential feedstock for mannooligosaccharides (MOS) production. MOS can be used in nutraceutical products for humans/animals or added to instant coffee, increasing process yield and improving product health properties. The SCG was evaluated for MOS production by steam pretreatment and enzymatic hydrolysis with a recombinant mannanase and a commercial cellulase cocktail (Acremonium, Bioshigen Co. Ltd, Japan). The mannanase was produced using a recombinant strain of Yarrowia lipolytica, used to produce and secrete endo-1,4-β,D-mannanase from Aspergillus aculeatus in bioreactor cultures. Endo-1,4-β,D-mannanase was produced with an activity of 183.5 U/mL and 0.23 mg protein/mL. The enzyme had an optimum temperature of 80 °C, and the activity in the supernatant was improved by 150 % by supplementation with 0.2 % sodium benzoate and 35 % sorbitol as a preservative and stabiliser, respectively. The steam pretreatment of SCG improved the enzymatic digestibility of SCG, thus reducing the required enzyme dosage for MOS release. Combined enzymatic hydrolysis of untreated or steam-pretreated (150, 190 and 200 °C for 10 min) SCG with mannanase and cellulase cocktail resulted in 36–57 % (based on mannan content) of MOS production with a degree of polymerization of up to 6. The untreated material required at least 1 % of both mannanase and cellulase loading. The optimum mannanase and cellulase loadings for pretreated SCG hydrolysis were between 0.3 and 1 and 0.4 and 0.8 %, respectively. Statistical analysis suggested additive effect between cellulase cocktail and mannanase on MOS release, with no indication of synergism observed.

Conversion of sugarcane bagasse to bioethanol is hindered due to intrinsic biomass recalcitrance that is related to its chemical composition and physicochemical properties. Furthermore, biomass heterogeneity interferes with process effectiveness. To compare sugarcane culm recalcitrance epi-dermis, internode and node fractions were individually investigated by acid pretreatment, enzymatic hydrolysis and thermal analysis/degradation. The epidermis fraction was found to be the largest mass fraction of the sucrose-free sugarcane culm followed by the epidermis-free internode and node fractions, comprising 65, 19 and 15.5 %, respectively. In comparison to the internode and node acid pretreatment solubilized a lower level of xylose from the epidermis resulting in higher mass recovery of water-insoluble solids (WIS) demonstrating its higher resistance to acid pretreatment. Enzymatic digestion showed that the epidermis is least susceptible to hydrolysis followed by the node and internode: 18.6, 56.5 and 75.9 %, respectively. In agreement with the enzymatic hydrolysis yield the internal/external surface area was lower for the epidermis than for the node and internode. Scanning electron microsco-py (SEM) showed the epidermis exhibited less structural damage after enzymatic hydrolysis. Moreover, the epidermis required a higher start temperature for degradation (330 °C) and exhibited a higher heating value (4,236 cal/g). The inter-node and node required a degradation start temperature of 288 and 265 °C and had heating values of 4,098.9 and 3,998.76 cal/g, respectively. Taken together the results of this study demonstrate that the epidermis is more resistant to pretreatment, to thermal and enzyme degradation than are the internode and node. The separation of the epidermis from the culm could provide a new perspective on proper use/ conversion of the most recalcitrant fraction of the sugarcane. In fact, separation of fractions decreases the biomass hetero-geneity with a positive impact on the effectiveness of the conversion process for selecting material more susceptive to pretreatment or optimizing the process for each fraction.

Instant coffee production generates large amounts of a waste known as spent coffee ground (SCG), which is rich in carbohydrate (∼50%), oil and aromatic compounds. Steam pretreatment by itself solubilised up to 10.7% of the SCG mass, which was recovered as soluble solids in
the liquor from pretreatment. The highest extent of soluble solid recovery from pretreatment itself (10.7%) was obtained
at 210°C/15 min, while milder conditions (150–190◦C/10–12 min) recovered 2.6% soluble solids. Mannanase (recombinant
strain of Yarrowia lipolytica) and cellulase (Acremonium, Bioshigen Co.Ltd, Japan) had an additive effect, with no indication
of synergism observed. The best combinations of mannanase/cellulase cocktail for enzymatic hydrolysis were 0.5/0.82% and
0.01/0.91% (w/w), releasing 20.6% (g per 100 g rawSCG) and 22.6% (g per 100 g pretreated SCG) of soluble solids fromuntreated
and pretreated SCG, respectively. By combining soluble solids recovered from steam pretreatment with the soluble products
from subsequent enzymatic hydrolysis, a yield of 27.65% (g per 100 g SCG) was achieved. The pretreatmentwaseffective in generating amaterialmoreamenable toenzymeaction, thus requiring reduced
enzyme dosages for subsequent hydrolysis compared with untreated SCG. The combined process showed significant technical
feasibility for re-use SCG andmay be considered to produce additional instant coffee product.

The alkaline extraction of hemicelluloses from hardwoods prior to pulping, for further conversion to value-added products, seems to be a promising pathway for paper mills to increase profit and improve sustainability. However, the amount of hemi-cellulose extracted will be limited by the requirement to maintain pulp quality and pulp yield in comparison to existing pulping processes. The effects of NaOH concentration, temperature, and time on hemicellulose extraction of Eucalyptus grandis were studied using a statistical experimental design. Extracted wood chips were subjected to kraft pulping to evaluate the effect of the extraction on cooking chemicals, pulp quality, and handsheet paper strengths. The selective xylan recovery (12.4% dry mass) from E. grandis combined with low-cooking, active alkali charge, and less cooking time ad-vantaged the xylan extraction and subsequent modified kraft pulping process under the studied conditions. Pulp viscosity, breaking strength, and tensile index of handsheets were slightly improved.

Spent coffee ground (SCG) is the main residue generated during the production of instant coffee by thermal water extraction from roasted coffee beans. This waste is composed mainly of polysaccharides such as cellulose and galactomannans that are not solubilised during the extraction process, thus remaining as unextractable, insoluble solids. In this context, the application of an enzyme cocktail (mannanase, endoglucanase, exoglucanase, xylanase and pectinase) with more than one component that acts synergistically with each other is regarded as a promising strategy to solubilise/hydrolyse remaining solids, either to increase the soluble solids yield of instant coffee or for use as raw material in the production of bioethanol and food additives (mannitol). Wild fungi were isolated from both SCG and coffee beans and screened for enzyme production. The enzymes produced from the selected wild fungi and recombinant fungi were then evaluated for enzymatic hydrolysis of SCG, in comparison to commercial enzyme preparations. Out of the enzymes evaluated on SCG, the application of mannanase enzymes gave better yields than when only cellulase or xylanase was utilised for hydrolysis. The recombinant mannanase (Man1) provided the highest increments in soluble solids yield (17 %), even when compared with commercial preparations at the same protein concentration (0.5 mg/g SCG). The combination of Man1 with other enzyme activities revealed an additive effect on the hydrolysis yield, but not synergistic interaction, suggesting that the highest soluble solid yields was mainly due to the hydrolysis action of mannanase.

The alkaline and acid preextraction of giant bamboo (Bambusa balcooa Roxburgh), grown in South Africa, before kraft and soda-anthraquinone (AQ) pulp-ing was in focus and the effects of NaOH concentration, temperature, and time on hemicelluloses extraction were studied by means of a statistical experimental design. The NaOH concentration exerted the strongest influence, solubilizing up to 20.4% of the available xylan with 2.0 M NaOH at 90°C for 240 min. The optimal extraction condition was achieved with 1 M NaOH at 90°C for 240 min, in the course of which 13.6% of the xylan was removed; the screened pulp yield was 50.4% without viscosity reduction , whereas the burst index of the handsheets was even improved. In the case of dilute acid preextraction before soda-AQ pulping, 11.3% xylan could be removed without affecting the pulp quality. The viscosity of the pulp was even better than that of pulps from nonextracted bamboo. The biorefinery approach, alkaline preextraction in combination with kraft pulping, is superior to the approach, acid prehydrolysis/soda-AQ pulping.

The extraction of hemicelluloses, which otherwise would be wasted in the black liquor, can be integrated with chemical pulping processes in a biorefinery approach that will generate a sugar-rich feedstock for production of fuels and chemicals. Extractions of hemicelluloses from sugarcane bagasse under dilute sulphuric acid or mild alkaline conditions were performed, using a central composite experimental design. Selected solid residues obtained after dilute acid, hot water (zero acid) or mild alkaline pre-extractions were subjected to soda or soda-AQ pulping, and kraft pulping of the dilute acid pre-extracted solid residue was also performed. The integration of hemicelluloses pre-extraction by alkaline methods into a soda-AQ-based pulping process was preferred for sugarcane bagasse as feedstock, since it enabled xylan recovery of 69.1%, while providing pulps with superior tear strength and brightness.

Sugarcane bagasse hemicellulose was isolated in a one-step chemical extraction using hydrogen peroxide in alkaline media. The polysaccharide containing 80.9% xylose and small amounts of L-arabinose, 4-O-methyl-D-glucuronic acid and glucose, was hydrolyzed by crude enzymatic extracts from Thermoascus aurantiacus at 50°C. Conditions of enzymatic hydrolysis leading to the best yields of xylose and xylooligo-saccharides (DP 2-5) were investigated using substrate concentration in the range 0.5–3.5% (w/v), enzyme load 40–80 U/g of the substrate, and reaction time from 3 to 96 h, applying a 2 2 factorial design. The maximum conversion to xylooligosaccharides (37.1%) was obtained with 2.6% of substrate and xylanase load of 60 U/g. The predicted maximum yield of xylobiose by a polynomial model was 41.6%. Crude enzymatic extract of T. aurantiacus generate from sugarcane bagasse hemicellulose 39% of xylose, 59% of xylobiose, and 2% of other xylooligosaccharides.

A process has been elaborated for one-step low lignin content sugarcane bagasse hemicellulose extraction using alkaline solution of hydrogen peroxide. To maximize the hemicellulose yields several extraction conditions were examined applying the 2 4 factorial design: H 2 O 2 concentration from 2 to 6% (w/v), reaction time from 4 to 16 h, temperature from 20 to 60 • C, and magnesium sulfate absence or presence (0.5%, w/v). This approach allowed selection of conditions for the extraction of low and high lignin content hemicellulose. At midpoint the yield of hemicellulose was 94.5% with more than 88% of lignin removed. Lignin removal is suppressed at low extraction temperatures and in the absence of magnesium sulfate. Hemicellulose in 86% yield with low lignin content (5.9%) was obtained with 6% H 2 O 2 treatment for 4 h and 20 • C. This hemicellulose is much lighter in color than samples obtained at the midpoint condition and was found suitable for subsequent enzymatic hydrolysis.

Different strains of the thermophilic ascomycetous fungus Thermoascus aurantiacus have been reported in the literature to produce high levels of a variety of industrial interest enzymes (i.e. amylases, cellulases, pectinases and xylanases), which have been shown to be remarkably stable over a wide range of temperatures and appear to have tremendous commercial potential. Most studies on enzyme production by T. aurantiacus are carried out in chemically defined liquid medium, under conditions suitable for induction of a particular enzyme. A few studies have investigated the production of some enzymes by T. aurantiacus by solid-state fermentation, using lignocellulosic materials. The present review focuses on the enzymes produced by T. aurantiacus, their main kinetic parameters, and the effect of different culture conditions on production and enzyme activity. It also provides a view of the possible applications of T. aurantiacus enzymes, considering that this thermophilic fungus could comprise a potential source of thermostable enzymes.

Thermoascus aurantiacus is able to secrete most of the hemicellulolytic and cellulolytic enzymes. To establish the xylanase inducers of T. aurantiacus, the mycelia were first grown on glucose up until the end of the exponential growth phase, followed by washing and re-suspension in a basal medium without a carbon source. Pre-weighed amounts of xylose (final concentration of 3.5 mg/ml), xylobiose (7 mg/ml) and hydrolyzed xylan from sugarcane bagasse (HXSB) which contained xylose, xylobiose and xylotriose (6.8 mg/ml) were evaluated as inducers of xylanase. It was observed that xylose did not suppress enzyme induction of T. aurantiacus when used in low concentrations, regardless of whether it was inoculated with xylobiose. Xylobiose promoted fast enzyme production stopping after 10 h, even at a low consumption rate of the carbon source; therefore xylobiose appears to be the natural inducer of xylanase. In HXSB only a negligible xylanase activity was determined. Xylose present in HXSB was consumed within the first 10 h while xylobiose was partially hydrolyzed at a slow rate. The profile of a-arab-inofuranosidase induction was very similar in media induced with xylobiose or HXSB, but induction with xylose showed some positive effects as well. The production profile for the xylanase was accompanied by low levels of cellulolytic activity. In comparison, growth in HXSB resulted in different profiles of both xylanase and cellulase production, excluding the possibility of xylanase acting as endoglucanases.

There are many changes, both qualitative and quantitative, in euca-lypt waste during growth and fructification of Lentinula edodes. Wet chemical analysis and near-infrared (NIR) spectroscopy were used in conjunction with multivariate regression and principal components analysis to monitor biodegradation of eucalyptus waste during growth of several L. edodes strains. Weight and component losses of eucalypt residue after biodegra-dation by L. edodes strains were compared for periods of 1 to 5 mo. Decrease in cellulose, hemicellulose, and lignin contents occurred, however it was not concomitant. Measurement of lignin degradation by NIR and wet chemical analysis indicated its attack in the early stages of biodegradation. Selective lignin degradation by L. edodes was observed up to 2 mo of biodeg-radation for strains DEBIQ and FEB-14. One group of degraded substrate was identified based on the principal component analysis (PCA) of the data on their biodegradation time. Samples treated for 5 months by L. edodes strains (DEBIQ, UFV or FEB-14) differed from other, but no discrimination was observed among them. By the end of 5 mo, NIR analyses showed decrease of about 18–47% cellulose, 35–47% polyose and 39–60% lignin. These data were used for comparison with those obtained by wet chemical

Covalent lignin-carbohydrate linkages between lignin and carbohydrates have been suggested to be a major obstacle to complete delignification of biomass feedstocks during chemical processing, e.g. pulping or enzymatic hydrolysis of the biomass to release sugars for ethanol production. The current study is aimed at developing an understanding of the structural composition and the behaviour of lignin carbohydrate complexes (LCCs) in sugarcane bagasse, a potential raw material for bio-ethanol production. Hemicelluloses were pre-extracted prior to soda AQ pulping with the aim of preserving it for further use instead of losing it in the black liquor (Value Prior Pulping or VPP). LCCs from the hemicelluloses pre-extracted soda AQ pulps as well, as non-extracted materials i.e. controls, were isolated and quantified. Mainly, two types of LCCs were identified: the xylan-lignin and glucan-lignin-xylan complexes. FTIR and GPC analyses of LCC fractions confirmed that the ester bonds of LCCs wer...