Manuscrito finalizado
El artículo reporta los resultados obtenidos del capítulo 3.1. Evaluación de distitnos pretratamientos sobre la composición química del bagazo de caña de azúcar, y su posterior efecto en el rendimiento de hidrolisis y fermentación.
Sugar cane bagasse pretreatments analysed through
13C CPMAS NMR:
implications for enzymatic hydrolysis and fermentation
Hernandez C1, Ziarelli F4, Gaime I2, Farnet AM3, García G1, Alarcon E1*
1Instituto de Biotecnología y Ecología Aplicada (INBIOTECA), Universidad Veracruzana, Xalapa Veracruz, México.
2Equipe Ecotechnologies et Bioremediation, IMBE, UMR CNRS 7263, IRD 237, Faculté des Sciences et Techniques de St.
Jérôme, Aix Marseille Université, Marseille, France
3
Equipe Systèmes Microbiens IMBE, UMR CNRS 7263, IRD 237, Faculté des Sciences Techniques de St. Jérôme, Aix Marseille Université, Marseille, France
4Aix Marseille Université, Faculte ́ des Sciences et Techniques de Saint-Jérôme, Spectropole, PO box 512, Avenue Escadrille
Normandie Nie ́men, 13397 Marseille cedex 20, France
*Correspondence author [email protected]
Abstract
Alkalin and biological pretreatments to lignocellulose are commonly used for enhance enzymatic hydrolysis, and enhance ethanol production. It is known that those pretreatments change the fiber composition of lignocellulose (e.g. decreases lignin content); nonetheless, few attemps have been done to detail the chemical changes that occur when different pretreatments are used. In this study, we used 13C CPMAS NMR technology to describe changes in sugarcane bagasse (SCB) pretreated with NaOH and Ca(OH)2 solutions; and with Pycnoporus sanguineus
(Polyporaceae). We found changes in alkyl C, Carboxyl C, Aromatic C (tertiary, quaternary and p-hydroxyphenyl C), O-alkyl C, aminoacids, ergosterol and chitin contents, and cellulose crystallinity index (CI); when SCB was submitted to different pretreatments. Changes in the chemical composition of SCB, was after related to enzymatic hydrolysis by Principal Component Analysis. We found that P. sanguineus promotes better lignin decay, glucose release and hydrolysis yields than the others pretreatments; also, P. sanguineus increases the amount of aminoacids and ergosterol in SCB; meanwhile, NaOH increases cellulose crystallinity index. The hydrolizates were fermented with Saccharomyces cerevisiae by 96 h, and analized by HPLC. The initial composition of the hydrolyzates, i.e. glucose and saccharose contents (mg.ml-1), and the biomass production (cells.ml-1) were then related to the ethanol production and fermentation yields. We found that ethanol production and fermentation yield were negatively correlated with biomass production, but positively correlated with glucose comsumption and with P. sanguineus pretreatment. We conclude, that a biological pretreatment, using P. sanguineus in the conditions here decribed, is a good way to increase ethanol productivity.
Introduction
Sugar cane bagasse (SCB) is a promising source of prime matter for cellulosic bioethanol production (Cardona et al., 2010); however, lignin and hemicellulose content, and cellulose crystallinity index represent an obstacle for enzymatic hydrolysis. Lignin can adsorb cellulases and the crystallinity of cellulose difficults the enzymatic hydrolysis (Hall et al., 2010; Mussato et al., 2008). Thus, for an efficient lignocellulose hydrolysis it is necessary a pretreatment process for decreasing lignin, hemicellulose content and the cellulose-crystallinity index.
Some of the most widely used pretreatments includes alkali agents, like NaOH, Ca(OH)2 and KOH (Maryana et al., 2014). Alkali attacks lignin via ester-linkages saponification, between cellulose and hemicellulose with lignin (Sun and Cheng, 2002); however, can generate alkalin-cellulose by ion exchange (Bahar et al., 2009). In addition, the use of alkali agents for biomass delignification, generally do not generate nocive compounds for microorganisms, contrary when acids are used (Yemis and Maza, 2011).
In the other hand, biological pretreatments using living fungi have been probed that contribute to enhance enzymatic hydrolysis (Nazarpour et al., 2013). Fungi (mainly white-root fungi), can oxidize lignin via phenoloxidase enzymes (i.e. laccases, Mn-peroxidases, lignin peroxidases), but also produce cellulases and hemicellulases (e.g. xylanases), decreasing the polysaccharides stock and the bioethanol production potential. Also, fungi transform some lignocellulose material into fungal biomass, changing the whole feedstock material. The structural characterization of SCB fibers, pretreated with living fungi, is necessary in order to understand how fungal enzymes affects the fibers composition, and how this structural composition affects the hydrolysis/fermentation downstream processes. Classical methods for fibers analysis based in subsequent detergent hydrolysis (e.g. Van Soest and Wine, 1964) are not enough for explain the complex modifications of lignocellulose fibers by fungi or chemical pretreatments, at cell wall level. Thus the use of other techniques like near infrared spectroscopy FTIR and 13C solid-state nuclear magnetic resonance (NMR) are preferred for lignocellulose characterization.
13
C CPMAS NMR spectroscopy is a powerful technique of analysis, successfully used for soil litter (Alarcón-Gutiérrez et al., 2009), lignification process (Terashima et al., 1997) and the extent of condensation in lignin (Liitiä et al., 2002) characterization; and can be used for evaluate changes in fungal biomass (Friĉová and Koval’aková, 2013). Solid-state NMR provide a way to do chemical analysis in a native state, and is a remarkable choice for samples with restricted solubility, as residuals lignins, or when the physical structure of cellulose is studied (Maunu, 2002). So, this study was conducted in order to characterize SCB fibers after a biological pretreatment using the fungi Pycnoporus sanguineus, and two alkaline pretreatments, using NaOH and Ca(OH)2 by 13C CPMAS NMR spectroscopy. After that, pretreated SCB was hydrolized with Aspergillus niger cellulasesand fermented
with Saccharomyces cerevisiae, and the yields of both steps were related to its structural composition, using multivariate statistical methods.
Materials and methods
Sugarcane bagasse pretreatments
SCB from the sugarmill of Mahuixtlán, Veracruz, México; was subjected to two kind of delignification pretreatments. The first one using an alkalin solution (with NaOH or Ca(OH)2) at 5 % w/v, and autoclaving by 15 min at 120° C and 15 lb of preasure. Delignified SCB was washed three times with dH2O, in order to remove sodium or calcium salts, after that, was dried at 60° C by 24 h and ground prior to 13C CP MAS NMR analysis, and cellulase hydrolysis.
The other pretreatment was biological. A volume of basal medium (Eggert, 1996) with the following composition (per litre): 1 g of KH2PO4, 0.26 g of NaH2PO4, 0.317 g of (NH4)2SO4, 0.5 g of MgSO4 7H2O, 0.5 mg of CuSO4, 74 mg of CaCl2, 6 mg of ZnSO4, 5 mg of FeSO4, 5 mg of MnSO4, 1 mg of CoCl2, supplemented with yeast extract (1 g.L-1) as nutrient source and pH adjusted to 6 was added to 50 g of pre-dried sugar cane bagasse (SCB) until a final humidity of 80% v/w. The wet SCB was deposited into poly-paper plastic bags and sterilized by autoclaving (120°C:15 lb of presure) for 20 min. After cooling, three-agar squares 0.25 cm2 in size with reactivated mycelium (7 days of culture) were used as inoculum. The mesocom were incubated in darkness at 30°C in an environmental chamber (Binder, GmbH). SCB samples were analyzed by 13C CPMAS NMR methodology, in order to determine the substrate modification by P. sanguineus. Each pretreatment: NaOH 5%, Ca(OH)2 5% and
Pycnoporus sanguineus (solid-state fermentation) were replicated three times.
13
C CP MAS NMR analysis
Changes in chemical composition of SCB were followed via 13C CPMAS NMR in a Bruker Advance DSX 400 MHz spectrometer (Bruker, Madison, WI, USA). 100 mg of dried and ground SCB were placed in a zirconium rotor and spun at the Magic angle (54.44°) at 10 KHz. The 13C CPMAS NMR technique was performed with a ramped 1H pulse during a contact time of 3 ms and with 1H decoupling during the acquisition time to improve the resolution. Recording 4 K transients with a recycling delay of 2 s represented standard conditions to obtain a good signal-to-noise ratio.
Analysis of NMR spectra was performed using the MestReNova v10.0.2 software (Mestre Lab Research S. L 2015). Specific regions of NMR spectra were utilized as indicators of chemical shifts in SCB (Table 1), those regions allowed us to evaluate changes in SCB polysaccharides content, cellulose crystallinity, xylane and lignin contents; and in fungal biomass production (i.e. aminoacids, ergosterol, quitin).
Enzymatic hydrolysis
Pretreated SBC was ground, and 0.5 g was disposed in 25 ml (2 % w/v) of sodium-acetate buffer pH 4.8.
Aspergillus niger cellulases (USB Affimetrix, USA) were added at a concentration of 160 mg.ml-1, in order to achieve 3.5 FPU.ml-1 (previous enzyme characterization). The reaction mixture was incubated in crystal flasks (250 ml) in an environmental chamber (Binder GmbH) at 50° C and 150 rpm. Enzymatic hydrolysis had a duration of 96 h, and each 24 hours a sample was taken for determining: cellulase activity (FPU), total reducing sugars; and glucose, xylose, arabinose, and mannose contents.
After 96 hours, the hydrolysis yield (Hy) was calculated according to the following ecuation (Maeda et
Table 1. Regions of 13C CPMAS NMR spectra utilized for assess chemical shifts in SCB
Component Position Reference
Alquil-C region 0-45 ppm
Aminoacids carbon γ 16.1-24.7 ppm Straus et al., (1997)
Aminoacids carbon β 28-37.6 ppm Straus et al., (1997)
CH3 of quitin 23.32 ppm Van de Velde and Kiekens (2004)
Ergosterol and derivates C4, C24, C20, C13, C10
37.05-42.85 ppm Yue et al., 2001; Wang et al., 2008; Toh
Choon et al., 2012
O-alquil C Region 45-110 ppm
Cellulose crystallinity Is calculated by dividing the
total area of the crystalline peak (87 to 93 ppm) by the total area assigned to the C4 peak (80 – 93 ppm).
Park et al., (2010)
Aminoacids carbón α 53.5-61.3 ppm Straus et al., (1997)
Aminoacids carbon δ 47.5-49.2 Straus et al., (1997)
Xylane C4 81.2 – 81.7 ppm Wickholm et al., (1998)
Hult et al., (2000)
Aromatic C Region 110 – 160 ppm
Aromatic tertiary C 110-123 ppm Hallac et al., (2009)
Aromatic quaternary C 123-160 ppm Hallac et al., (2009)
p-hydroxyphenyl 157-162 ppm Hallac et al., (2009)
Carboxil-C Region 160-190 ppm
Quitin C=O 173.7 ppm Van de Velde y Kiekens (2004)
Carbon of COOH from
aminoacids
al., 2011):
𝐻𝑦 =𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑 (𝑚𝑔/𝑚𝑙)
𝑆𝐶 × 𝐶𝐶 × 1.1
Where:
SC = substrate content (mg.ml-1)
CC = holocellulose content in substratum (g.g-1)
1.11 = Is the correction factor derivated of the addition of water molecules into anhidroglucose residues
Fermentation
After enzymatic hydrolysis, the volume was diluted with dH2O (1:4) and 0.34 mg.ml-1 of dry cells of
Saccharomyces cerevisiae were used as inoculum, according to Li et al (2011). The crystal flasks were incubated at 30 °C and 150 rpm by 96 hours. Each 24 hours a sample was taken for determining the glucose, xylose, mannose, arabinose and ethanol content; biomass was estimated by direct counting with a Neubauer chamber. Finalizing the incubation time, the fermentation effiency was calculated by the following ecuation (Yadav et al., 2011):
𝐹𝑒𝑟𝑚𝑒𝑛𝑡𝑎𝑡𝑖𝑜𝑛 𝑒𝑓𝑖𝑒𝑛𝑐𝑦 = 𝑃𝑟𝑎𝑐𝑡𝑖𝑐𝑎𝑙 𝑦𝑖𝑒𝑙𝑑
𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑦𝑖𝑒𝑙𝑑 𝑥 100
Where:
Practical yield = ethanol produced
Theoretical yield = 0.511 per gram of glucose consumed
Analytical methods
Cellulase activity was measured calculating the Filter Paper Units of 0.5 ml of solution, according to Ghose (1987). For this, the glucose equivalents released by diferent enzyme dilutions from a strip of 6 x 1 cm (50 mg) of filter paper (Whatman No. 1) were determining by the method of dinitrosalicilic acid (DNS; Miller, 1959), and used to estimate the enzyme needed to release 2.0 mg of glucose in 60 min, at 50°C. The reaction mixture employed was as follows: 0.5 ml of enzyme dilution + 1 ml of sodium- acetate buffer pH 4.8 + strip of filter paper. After the incubation time, the reaction was stoped with 3 ml of DNS reagent, boiled for 5 min and cooled in an ice bath by 20 min. 200 µl of the reaction mixture were diluted in 2.5 ml of dH2O and read at 540 nm. With those data FPU.ml was calculated following the next ecuation:
𝐹𝑃𝑈 = 0.37
𝐸𝑛𝑧𝑦𝑚𝑒 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑡𝑜 𝑟𝑒𝑙𝑒𝑎𝑠𝑒 2.0 𝑚𝑔 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒𝑢𝑛𝑖𝑡𝑠. 𝑚𝑙
Monomeric sugars, i.e. glucose, xylose, arabinose, mannose and ethanol, were quantified using High Performance Liquid Chromatography (Aliance Waters e2695), with a column HPX-87H Aminex BioRad, at 40° C and with a flux of 0.8 ml.min-1.
Statistical analysis
The data matrix of chemical composition of the different SCB submitted to the different pretreatments used, was analysed with the data of the hydrolysis products and yield, and with the data of the fermentation products; using a Canonical Correspondence Analysis (CCA), where the qualitative variables were the different pretreatments used. This analysis allowed us to found relations between the chemical composition of SCB obtained from 13C CPMAS NMR, and the products obtained in the downstream process.
Results
Chemical shifts in SCB submitted to different pretreatments
The chemical composition of the SCB changed after chemical and biological pretreatments. O-alkyl C signal, corresponds to the carbohydrate (i.e. holocellulose) content of SCB; this signal indicates, that the original composition of SCB have 79.76% of holocellulose, and after an alkalin pretreatment with NaOH the proportion increases to 88.86%, and to 84.13% when Ca(OH)2 was used. Contrary, the biological pretreatment decreases the amount of holocellulose to 50.65%. Aromatic C region, which corresponds to lignin, had an original proportion of 12.87%, and the pretreatments decreses this value as follows: Ca(OH)2 to 10.26%, NaOH to 7.03% and P. sanguineus to 6.53%. The value of the regions Carboxyl C and Alkyl C, are associated with proteins and lipids (Table 1), and these values were very low in the original composition of SCB: 3.57% and 2.45%, respectively. Alkalin pretreatments disminish the amount of Carboxyl C to 1.48% (NaOH) and 1.60% (Ca(OH)2), and in general, did not affected Alkyl C region. In the other hand, the pretreatment with P. sanguineus strongly increases both regions, Carboxyl C increases until 9.31%, and Alkyl C until 33.49% (Figure 1a).
Signals corresponding to Cα, Cβ and Cδ of aminoacids, increased in the treatments with P. sanguineus from 5.61% to 18.38%, as well as signals from chitin and Cγ of aminoacids, from 3.47% to 8.49%; alkalin treatments diminish or did not changed those signals (Figure 1b). Ergosterol signal was
near to 0 in the original composition of SCB, and remained in the same way after alkalin pretreatments. However, when P. sanguineus was employed, ergosterol increased from 0.24% to 4.33%.
The proportion of amorphous cellulose and crystalline cellulose, estimated by the signal position of the C4 of pyrano-glucose (Figure 1c), indicates that SCB has an original crystallinity index (CI) of 0.29. This value, changed a little according to the pretreatments employed: SCB pretreated with NaOH increased its CI to 0.32; decreased to 0.27 when Ca(OH)2 was used, and remained unchanged in P.
sanguineus pretreatment. Finally, the signals of aromaric carbons decreased in all treatments, been P. sanguineus the treatment that reached the highest decrease of aromatic carbons, near followed by SCB pretreated with NaOH, and third, the SCB pretreated with Ca(OH)2.
Figure 1. Chemical composition of SCB after an alkalin or biological pretreatment. a) Major components of NMR spectra, related to the presence of organic macromolecules (i.e. lipids, proteins, carbohydrates and aromatic compounds). b) changes in signals associated to the development of the fungal biomass, i.e. aminoacids, chitin and ergosterol. c) Changes in C4 of glucose and xylose, which can be associated to an increase or decrease of crystalline/amorphous cellulose, and xylene. d) Changes in the carbon composition of lignin.
Hydrolysis step
Cellulase activity decreases rapidly in the first 24 hours, in all treatments; meanwhile, total sugars content increases in these 24 hours, and after that remained almost unchanged (Figure 2). The area under the curve of the FPU activities, showed that when SCB pretreated with P. sanguineus (99.21 FPU in 96 h) and NaOH (115 total FPU in 96 h) were hydrolized, the enzymatic stability was higher. Contrary, less FPU activity was registred in treatments with SCB pretreated with Ca(OH)2 (56.91 total FPU in 96 h) and SCB no-treated (57.33 total FPU in 96 hours).
Figure 2. Cellulase (FPU.ml-1) kinetics in 96 h SCB hydrolysis (right Y axis). In color, kinetics of reducing sugars content (mg.ml-1) (Left Y axis). Middle points indicates average values, and bars the standard error, n = 3.
standard error, n = 3.
Similar to total reducing sugars release, glucose production was faster in the first 24 h of hydrolysis. SCB pretreated with P. sanguineus produced the highest amount of glucose (29.76 ± 2.2 mg.ml-1), followed by SCB pretreated with Ca(OH)2 (28.19 ± 0.45 mg.ml-1), NaOH (23.84 ± 2.0 mg.ml-1), and finally, the no-treated SCB (9.95 ± 15.93 mg.ml-1) (Figure 3). Saccharose content, decreases slightly during the SCB hydrolysis in all treatments (Figure 4); reaching a final concentration of 4.38 ± 0.36 mg.ml-1, 4.26 ± 0.4 mg.ml-1, 3.66 ± 0.3 mg.ml-1 and 1.48 ± 2.4 mg.ml-1; when SCB pretreated with Ca(OH)2, P. sanguineus, NaOH and no-treated were used, respectively (Figure 4).
Figure 4. Saccharose (mg.ml-1) content during enzymatic hydrolysis (96 h). Middle points indicates average values, and bars the standard error, n = 3.
With data of glucose production, and the holocellulose initial content, we estimated the hydrolysis yield. Thus, the highest hydrolysis yield was achieved in the treatments when SCB pretreated with P. sanguineus, followed by the SCB pretreated with Ca(OH)2, NaOH and finally, the no-treated SCB.
Fermentation step
In the fermentation step, the hydrolyzate was diluted 1:6 with dH2O, the differences in the hydrolysis yield provoked differences in the initial glucose content of the fermentation cultures (Figure 5). Glucose decreased near to 0 mg.ml-1 in all treatments, after 48 h of culturing with S. cerevisiae; menwhile, saccharose was metabolized slower (Figure 6). On the other hand, S. cerevisiae biomass (Figure 8) and ethanol, increased until 96 h, the hydrolizate of SCB pretreated with NaOH (3.26 ± 0.3 mg-ml-1) and P. sanguineus (2.99 ± 0.2 mg.ml-1), were those who produced more ethanol; followed by no-treated (2.11 ± 0.8 mg.ml-1) and Ca(OH)2 (1.50 ± 0.3 mg.ml-1) (Figure 7).
The highest fermentation yield was attained from the hydrolizate of SCB pretreated with P. sanguineus, followed by no-treated SCB, NaOH, and finally, Ca(OH)2.
Figure 5. Glucose (mg.ml-1
) content during fermentation (96 h). Middle points indicates average values, and bars the standard error, n = 3.
Figure 6. Saccharose (mg.ml-1
) content during fermentation (96 h). Middle points indicates average values, and bars the standard error, n = 3.
Figure 7. Ethanol (mg.ml-1
) content during fermentation (96 h). Middle points indicates average values and bars the standard error, n = 3.
Figure 8. Cells of Saccharomyces cerevisiae (cells.ml-1) content during fermentation (96 h). Middle points indicates average values, and bars the standard error, n = 3.
Multivariate analysis
Chemical composition of SCB pretreated with P. sanguineus or alkali solutions, affected the hydrolysis yield and the hydrolysis products obtained. Glucose released was positive correlated with Alkyl C, Carboxyl C, aminoácids content, chitin and ergosterol; these variables positive affected the hydrolysis yield too (Table 2).
Table 2. Pearson correlations (r) between chemical components of pretreated SCB and hydrolysis products/enzymatic activity.
Compounds Cellulase stabillity Glucose released Hydrolysis yield Saccharose final content
Alkyl C -0.592 0.746 0.923 0.435 Carboxyl C -0.500 0.545 0.786 0.158 Aromatic C 0.117 -0.653 -0.668 -0.739 O-alkyl C 0.597 -0.634 -0.847 -0.254 aaƴ+chitin -0.478 0.489 0.742 0.087 aaCβδα -0.675 0.797 0.950 0.483 Ergosterol -0.599 0.762 0.932 0.459 Xylane C4 0.497 -0.593 -0.823 -0.232 Cellulose crystallinity 0.793 -0.311 -0.268 -0.036 Tertiary aromatic C 0.257 -0.757 -0.740 -0.833 Quaternary aromatic C 0.057 -0.604 -0.632 -0.694 p-hydroxyphenyl aromatic C 0.247 -0.736 -0.633 -0.908
Meanwhile, cellulose crystallinity, Aromatic C, and tertiary, quaternary and p-hydroxyphenyl aromatic C, negatively affected glucose release and hydrolysis yield. Cellulase stability was positive correlated with O-alkyl C, Xylane C4 and Cellulose crystallinity; and negative correlated with Alkyl C, Carboxyl C, carbon aminoacids, chitin and ergosterol. Saccharose was positive correlated with the variables associated to the presence of fungal biomass, and negatively correlated with lignin compounds (aromatic carbons) (Table 2).
PCA Biplot, showed, that the treatment which promotes more glucose and hydrolysis yield was P. sanguineus. Pretreatment with NaOH and Ca(OH)2 (less effect), increases cellulose crystallinity, but increases O-alkyl region and the cellulase stability. No-treated SCB had the highest amounts of liginin compounds, and was negatively associated with glucose release and hydrolysis yield (Figure 9).
Figure 9. PCA biplot showed the relation between chemical composition of SCB and hydrolysis variables (red lines), with the pretreatments used (yellow circles).
In the same way, hydrolizates composition affected the fermentation yield and the ethanol production. Biomass production was positive correlated with glucose consumed, but negative related to fermentation yield and ethanol production (Table 3). It was not observed strong relations of the variables measured with the ethanol production and fermentation yields. Nevertheless, PCA biplot indicates that the hydolyzed composition that comes from SCB pretreated with P. sanguineus, reached the best ethanol and fermentations yields (Figure 10). Contrary, the worst hydrolysate composition for ethanol production was which used SCB pretreated with Ca(OH)2.
Obs1 Obs2 Obs3 Obs4 Pretreatment- NaOH Pretreatment- Ca(OH)2 Pretreatment-P. sanguineus Pretreatment- Control Alkyl C Carboxyl C Aromatic C O-alkyl C aaƴ+chitin aaCβδα Ergosterol Xylane C4 Cellulose crystallinity Tertiary aromatic C Quaternary
aromatic C p-hydroxyphenyl aromatic C Cellulase stabillity Glucose released Hydrolysis yield Saccharose final content -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 -3 -2.5 -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 F2 (23. 14 %) F1 (64.95 %) Biplot (ejes F1 y F2: 88.09 %)
Table 3. Pearson correlations (r) between hydrolizates composition and fermentation products.
Variables Glucose consumed Fermentation yield Ethanol production Biomass production
Cellulase stabillity -0.165 0.025 0.373 0.076
Glucose released 0.221 0.129 0.061 -0.118
Hydrolysis yield -0.064 0.333 0.204 -0.370
Saccharose final content 0.510 -0.053 0.104 0.172
Biomass production 0.77 -0.65 -0.33 1
Figure 10. PCA biplot showed the relation between hydrolysate composition and fermentation variables (red lines), with the pretreatments used (yellow circles).
Discussion
We observed, that the pretreatments evaluated, changed the chemical composition of SCB, comparing with control. All treatments were able to decrease the aromatic C content, but the highest lignin decay was achieved with P. sanguineus; this treatment, enriched the aminoacid, ergosterol and chitin amount of SCB, but decreased the carbohydrate proportion. Thus, P. sanguineus transformed a portion of the SCB into fungal biomass, as was hypothesized at beginning; however, this fungal biomass gain does not interfer with the enzymatic hydrolysis. In fact, the highest hydrolysis yield was obtained with SCB
Obs1 Obs2 Obs3 Obs4 Obs5 Obs6 Obs7 Obs8 Obs9 Obs10 Obs11 Obs12 Pretreatment- NaOH Pretreatment- Ca(OH)2 Pretreatment-P. sanguineus Pretreatment- Control
Cellulase stabillity Glucose released
Hydrolysis yield Saccharose final content Glucosa consumida Fermentation yield Ethanol production Biomass production -3 -2 -1 0 1 2 3 -4 -3 -2 -1 0 1 2 3 4 5 F2 (36. 96 %) F1 (43.29 %) Biplot (ejes F1 y F2: 80.25 %)
pretreated with P. sanguineus.
The effect of proteins or lipids on cellulase catalysis is not well documented, but it is known that some lipids can act like surfactans, and avoid the absortion of cellulases by cellulose, increasing the hydrolysis yield (Helle et al., 1993; Eriksson et al., 2002). The same rol can be performed by some proteins, like bovine serum albumin (Yang and Wyman, 2006). Thus, the enrichment of proteins and ergosterol by P. sanguineus pretreatment, could be decreasing the cellulase absortion in SCB fibers, and improving the enzymatic hydrolysis. The decay in aromatic C and O-alkyl C regions in P. sanguineus pretreatments,