Organosolv Pretreatment of Sugar Cane Bagasse for Bioethanol

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Organosolv pretreatment of sugarcane bagasse for bioethanol production RULY TERAN HILARES, Mateus Pereira Swerts, Muhammad Ajaz Ahmed, Lucas Ramos, Silvio Silvério da Silva, and Julio Cesar Santos Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b00079 • Publication Date (Web): 20 Mar 2017 Downloaded from http://pubs.acs.org on March 21, 2017

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Industrial & Engineering Chemistry Research

Organosolv pretreatment of sugarcane bagasse for bioethanol production Ruly Terán Hilares a,*, Mateus Pereira Swerts a, Muhammad Ajaz Ahmed b, Lucas Ramos a, Silvio Silvério da Silva a, Júlio César Santos a a

Department of Biotechnology, Engineering School of Lorena, University of São Paulo, CEP 12602-810, Brazil b

Department of Civil and Environmental Engineering, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea *Corresponding author: ([email protected]; Tel.: +55 12 31595313; Fax: +55 12 31533006)

Abstract: A lowering in glycerol price has been observed last years, considering it is a by-product of biodiesel production. This fact has motivated studies about alternative uses for this compound, as in pretreatment of lignocellulosic biomass to produce ethanol, another biofuel of interest. In this context, glycerol acid-pretreatment of sugarcane bagasse (SCB) was evaluated in terms of temperature (80-120°C), solid loading (2.5-7.5%), glycerol (85-95%) and acid concentration (0.5-3%), and reaction time (10-60min), using a 25-1 factorial design. The cellulose digestibility was further optimized for the variables: temperature (120-130°C), acid/SCB mass ratio (5-15%) and time (20-60 min). The optimal condition was obtained at 130 °C of temperature, 15% of acid/SCB mass ratio and 57.5 min reaction time, yielding a 68.5% of digestibility.

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Moreover, a considerable amount of ethanol 0.38 g/g biomass (0.57 g/L.h of productivity) was achieved using Saccharomyces cerevisiae also shows the efficacy of this pretreatment. Keywords:

Organosolv

pretreatment;

glycerol;

sugarcane

bagasse;

enzymatic

hydrolysis; ethanol production 1.

Introduction Fossil fuels are overwhelming energy sources in the current global energy

scenario. However, their utilization has led to the serious environmental effects including but not limited to air pollution, contamination of water, greenhouse gases emissions, smog in urban areas and global warming. From these, greenhouse gases emissions are mainly due to the accumulation of carbon dioxide in the atmosphere and values higher than 400 ppm have been currently recorded, according to The Global Greenhouse Gas Reference Network, from NOAA's Earth System Research Laboratory of Boulder (Colorado, USA) 1. Therefore, it is an urgent need to substitute these conventional energy sources in a sustained way. Nowadays, ethanol is extensively produced from sugarcane in Brazil and corn in USA 2, but its contribution to meet the huge and growing energy demand can increase only by using other raw materials of larger availability in different regions of Earth. In this way, lignocellulosic ethanol has attracted more interest due to large quantity of agro industrial (sugarcane bagasse, rice straw, corn stover, etc.) or forest residues produced annually worldwide. However, these polysaccharides are joined together in a complex structure enough to make them inert against any biochemical action. This feature thus turns pretreatment methods as a prerequisite to make these sugars available for sub sequent processing. Pretreatment step is considered as one bottleneck for the commercialization of


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lignocellulosic biofuels. In this regard, different pretreatment techniques have been studied e.g. steam explosion 3, extrusion 4, hydrodynamic cavitation 5, ionic liquid 6, organosolv 7 and hydrothermal pretreatment 8 . Among from these strategies, organosolv pretreatment seems a potential scheme to enhance enzymatic digestibility of lignocelluloses with an improved lignin removal efficiency 9. It can employ a variety of organic solvents and their solution including low boiling liquids either (e.g. ethanol or methanol) or high boiling-point (e.g. ethylene glycol or glycerol) in acidic or non-acidic conditions 10. Glycerol is a product released during the transesterification of alcohols (methanol or ethanol) and triacylglycerols in the biodiesel production process 11,12. This by-product is a potential source for the production of various bioproducts 13. Also, it can be used as a pretreatment reagent because it produces significant changes in the biomass structure, by breaking functional groups and bonds

14,15

. Usually, it is used in acidic or alkaline

conditions to enhance the efficiency at relatively high temperatures (180-200°C) as reported for wheat straw 14, sugarcane bagasse 16 and rice straw 17. However, crude glycerol can be used for the pretreatment of lignocelluloses at rather low temperatures even in non-pressurized systems. So, to get benefit from glycerol in a context of integrated biorefineries, this work deals with its use for pretreatment of SCB in acidic medium. The objective was to use, in pretreatment step, a glycerol aqueous solution with a concentration range possible to be observed in crude glycerol produced in some biodiesel plants, that can reach more than 90 wt%

18

, or at

least possible to be obtained without extensive purification steps. In this way, considering the limit in boiling point of the solution in non-pressurized systems, experiments were performed in low temperatures. A sequential strategy of experimental design (25-1 followed by a central composite rotational design - CCRD) was used to



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optimize the process conditions and the fermentability of the SCB enzymatic hydrolyzed into ethanol was further assessed by Saccharomyces cerevisiae. 2.

Materials and methods

2.1. Materials Sugarcane bagasse (SCB) was obtained from the Usina São Francisco (Sertãozinho – SP, Brazil). It was washed and dried and following milled using a Benedetti 270 hammermill (Mill Benedetti Ltda, Pinhal-SP, Brazil). Average particle size between 0.62-1.24 mm, as classified using standard Tyler sieves, was used in the experiments. Main components of SCB such as cellulose (41.2%), hemicellulose (29.2%) and lignin (21.27%) were determined according to method described by Sluiter et al. 19. Glycerol was purchased from Sigma-Aldrich (Saint Louis, Missouri, USA). 2.2. Pretreatment of sugarcane bagasse Initial tests were carried out in order to evaluate the potential use of glycerol in the sulfuric acid-containing medium for pretreatment of SCB at atmospheric pressure. These experiments were performed during 10 min in Erlenmeyer flasks (250 ml) with 100 g of total reaction medium at temperature of 121°C using a heating plate. The reaction medium was composed by 5wt% of solid loading, 0.5 wt% of sulfuric acid in water or aqueous solution of glycerol (95 wt%). After reaction, the biomass was vacuum filtered and the liquid containing glycerol was stored in plastic containers. The solid fraction was washed and used wash water was mixed with liquid previously stored in the containers; after this, the solid was washed with 2 volume of warm water. The SCB composition was analyzed according to Sluiter et al.

19

and enzymatic hydrolysis

was carried out in the pretreated material according to section 2.3. Following, statistical screening was performed to sort out the more influential pretreatment variables. A 25-1 factorial design with 16 runs and triplicate at center points


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was used for the evaluation of: glycerol concentration (85 – 95 wt %), solids loading (2.5 – 7.5 wt %), sulfuric acid concentration (0.5 – 3 wt %), temperature (80 – 120 °C) and reaction time (10 – 60 min). The cellulose hydrolysis yield (enzymatic digestibility) was considered as a response in this step of the work. Hydrolysis yield was calculated as the ratio of the total mass of glucose released after enzymatic hydrolysis step and the theoretical value that could be obtained considering total cellulose fraction present in the material. Considering the results of the 25-1factorial design, a new central composite rotatable design (CCRD) was performed further to evaluate the pretreatment efficacy. The effects of the variables such as temperature (116.6 - 133.4 °C), acid/SCB mass ratio (1.6-18.4 wt %) and reaction time (6.4-73.6 min) were studied with a glycerol concentration of 90 wt % and a solids loading of 7.5 wt %. For these experiments, cellulose hydrolysis yield and solids composition (cellulose, hemicellulose and lignin) were considered as responses for optimization of the process. Statistical analysis was performed aided by the software STATISTICA version 8 (StatSoft, Inc., Tulsa, USA). 2.3. Enzymatic hydrolysis Enzymatic hydrolysis was performed in Erlenmeyer flasks (50 ml) with total reaction medium of 15 ml, composed by 2 wt % of pretreated biomass, citrate buffer (50 mM, pH=4.5) and 10 FPU/g of pretreated SCB (40.75 mg of protein/g of pretreated SCB) of commercial Dyadic® Cellulases (Dyadic International, Inc, USA). Hydrolysis were carried out in rotary shaker Quimis Q816M20 (Quimis, SP, Brazil) at 150 rpm and 50°C by 24 h. After 24 h, the enzymes were inactivated by thermal treatment (100 °C, 5 min), centrifuged and the sugar content was analyzed in supernatant by High Performance Liquid Chromatography (HPLC), according to Ahmed et al. 20. 2.4. Ethanol production



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Microorganism and inoculum Saccharomyces cerevisiae 405 was obtained from the strain stock available in the Microbiology and Bioprocess Group (GMBio) of the Engineering School of LorenaUniversity of Sao Paulo (EEL-USP), Brazil. For the inoculum preparation, a loopful of stock culture was transferred to 125 ml Erlenmeyer flasks containing 50 ml of medium composed by 30 g/L of glucose, 5 g/L of peptone, 3 g/L of yeast extract and 0.25 g/L of ammonium phosphate. Inoculum was growth by 24 h, 30 °C and 150 rpm using a rotary shaker New Brunswick™ Innova® 40/40R (Hamburg, Germany). After 24h, cells were recovery by centrifugation (3000 xg) and washed twice with sterile distillate water. A suspension of cells was added in fermentation flasks aiming to obtain 1 g/L of initial biomass concentration in the process. Fermentation process Fermentation was carried out in 50 ml Erlenmeyer flasks with 20 ml of medium composed by SCB enzymatic hydrolysate (18 g/L of initial glucose concentration), obtained from enzymatic hydrolysis of SCB pretreated at the optimized conditions. The medium was supplemented with 1 g/L of peptone, 1g/L of yeast extract, 1g/L of ammonium phosphate, 0.5 g/L of potassium phosphate, 0.5 g/L of magnesium sulphate and 0.5 g/L of manganese sulphate. Fermentation was carried out during 24 h at 30 °C and 100 rpm using a rotary shaker New Brunswick™ Innova® 40/40R (Hamburg, Germany), and samples were taken periodically for sugar, ethanol and biomass analysis. Sugar and ethanol were analyzed using High Performance Liquid Chromatography (HPLC), according to Ahmed et al.

20

. Concentration of biomass was determined by

turbidimetry using spectrophotometer Beckman DU 640B (Fullerton, USA) at wavelength of 600 nm and correlated with the dry weight of cells (g/L) through a calibration curve.


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3.

Result and discussion

3.1. Assessment of glycerol for SCB pretreatment Preliminary experiments were conducted in order to evaluate the effect of glycerol on the enzymatic digestibility and solid composition of SCB (Figure 1). As observed, the hydrolysis of cellulose obtained was variable between 11% (untreated material) to 42%, this last value observed in SCB pretreated at 121°C in medium content glycerol. As it is also shown, the cellulose hydrolysis yield obtained using glycerol-acid pretreated SCB was higher than the one obtained using diluted acid pretreated biomass. These results shown the potential effect of glycerol on the biomass improving the efficiency of hydrolysis, that is according to previous reports in the literature

16,21

.

However, despite of the higher enzymatic digestibility observed using glycerol, no great difference was observed in the solid composition (mainly in the cellulose content) of SCB pretreated with and without this solvent (Fig. 1). This indicates that glycerol effect can be related to modifications in material structure (increase in the porosity), instead of only compositional changes, easing the access and action of enzyme on the cellulosic fraction 21. Considering this preliminary result, experiments were conducted to screen out the more influential variables of glycerol pretreatment using a 25-1 factorial design. The results for the responses hydrolysis yield and solids composition, are summarized in Table 1. As shown in the Table, the composition of SCB in glycerol-acidified medium was modified, more likely due to the hydrolysis of hemicelluloses and, consequently, an increase in cellulose fraction percentage. For example, hemicellulose content is lower (6%) at run 4 when harsh conditions were applied and the corresponding cellulosic fraction was increased (68%). However, the highest hydrolysis yield of 65.6% was achieved at run 10, when a relatively higher concentration of acid and glycerol were



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used at 120 °C of temperature. In a previous report of Zhang et al.16, the efficiency of glycerol-acid pretreatment was also demonstrated. In that study, the cellulose contents in the biomass increased from 42.9% to 60%, the hemicellulose was reduced from 22.8% to 8% and lignin was kept around 26%. In our work, similar behaviors in the composition were observed after glycerol-acid pretreatment, mainly considering the carbohydrate fractions. In order to evaluate the pretreatment efficacy, an analysis of main effects of the studied variables was performed (Table 2). As depicted in the table, only the temperature had significant positive effect (p-value < 0.05) on the cellulose contents. It has also the similar effect for hemicellulose but with a negative trend. Among from other variables, time had a significant and negative effect (p-value < 0.05) and glycerol concentration had a significant positive effect (p-value < 0.1) on hemicellulose content. No variable had significant influence on lignin content in pretreated material (p-value > 0.1 for all effects). The importance of effects on the response enzymatic hydrolysis was in the following order: temperature > acid concentration > time of reaction > solid loading > glycerol concentration. However, only temperature and acid concentrations showed significant effect (p-value < 0.05), although p-value for time was near 0.1. Considering the above results, temperature, time and acid/bagasse ratio were screened to design a central composite rotational design (CCRD). In this new design, temperature range was from 117-133°C, considering the positive effect of this variable showed in Table 2. Although the effect of acid concentration was positive, in the new experimental design its values was reduced aiming to use lower acid/bagasse mass ratio (that reached until 120% in experiments correspondent to Table 1), turning the global cost of the process more attractive. This variable was then evaluated as acid/bagasse mass ratio and the range was 1.59-18.41


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wt%. As the main effect of variable time was near the significance level of 90% (Table 2), it was included in the new design. The effect of glycerol variable was lower than other studied, and 90% of glycerol concentration was considered in the new design. Thus, to avoid reaching the boiling point of pretreatment liquid mixture (boiling point is 138 °C for 90% aqueous glycerol solution) and to keep a non-pressurized system, the maximum evaluated temperature was 133°C. Solid loading was fixed in 7.5% in order to use the total volume of reactor. 3.2. Optimization of glycerol-acid pretreatment conditions using CCRD The effects of screened variables on the response of enzymatic hydrolysis yield are shown in Table 3. As can be observed, cellulose hydrolysis yield reached a maximum value of 64.1%, at run 8. By comparing this result with that one obtained at run 10 in Table 1, the effect of temperature was confirmed, as an increase in about 10 °C of temperature was enough to result in enzymatic hydrolysis yield above 60% even using lower acid concentration in CCRD experiments compared to those used in 25-1 assays. This effect is probable due to structural changes (high temperatures and long times produce change in the crystallinity of cellulose), oxidation and cleavage of some intermolecular linkages by thermal effect

22

. These results support that acid-catalyzed

glycerol pretreatment is indeed effective even at mild thermal conditions to improve the hydrolysis yield 16,21. An ANOVA was performed for a quadratic model as a function of the studied variables (Eq. 1 and Table 4). The p-value of the model was lower than 0.0001, with Fvalue of 24.11, indicating that the model is significant at 95% of confidence level. Lack of fit was not significant and the coefficient of determination was 0.865, indicating the model can explain 86.5% of variability of the response variable in the studied range. The optimal pretreatment combination was found at 130°C, 15% of acid/SCB mass ratio



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and 57.5 min of reaction time, with a predicted value of 64.1 % of hydrolysis. It was further confirmed experimentally with a 69.1 ± 5.2 % hydrolysis yield, indicating that the model was adequate. Moreover, under this condition, solid recovery (ratio between SCB mass after and before pretreatment) was 63%, with a removal of cellulose, hemicellulose and lignin of 14%, 78% and 1.6%, respectively. ܻଶ = −27.2 + 0.19ܺଵ − 18.40ܺଶ + 0.29ܺଷ − 0.14ܺଶଶ + 0.18ܺଵ ܺଶ

Eq. (1)

Where Y2= Cellulose hydrolysis yield (%), X1=Temperature (°C), X2=Acid/SCB mass ratio (%), X3= Pretreatment time (min). As a general viewpoint, the obtained results showed the potential use of glycerol in the pretreatment process. Due to the effect of this kind of pretreatment, mainly the hemicellulosic fraction was removed. Although no high lignin removal was observed, enzymatic digestibility of pretreated SCB was higher perhaps due to an increase in the material porosity and so for enzyme-access. The low severity and non-pressurized pretreatment process is an attractive alternative and, in an integrated biorefinery, residual solid remaining after hydrolysis can be used as an energy source. 3.3. Fermentability of cellulosic hydrolysate In order to evaluate the fermentability of enzymatic hydrolysate of glycerolpretreated SCB by Saccharomyces cerevsiae, fermentation was carried out during 24 h (Figure 2). Complete sugar consumption was observed in 18 h of fermentation and the maximal ethanol production, 6.8 g/L, was achieved in 12h of process and was correspondent to 0.38 gp/gs of ethanol yield (efficiency of 75% considering a theoretical yield of 0.51 g/g) and 0.57 g/L.h of volumetric productivity. This result, although using non-optimized conditions with relation to the parameters as substrate and initial cells concentration, showed the potential of use of the obtained hydrolysate for ethanol production. In other literature work, e.g., Mesa et al. 23,


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using sugarcane bagasse pretreated with organosolv process (50% v/v aqueous ethanol solution, 1.25% w/w of sulfuric acid, 175 °C by 60 min), related a higher ethanol yield (92.76% based in the theoretical yield of 0.51 g/g) in fermentation using S. cerevisiae, but harsher pretreatment and different fermentation conditions were used. In other work, Phi Trinh et al.

24

reported fermentation of hydrolysate of rice straw pretreated

with organosolv process (glycerol with 70% of purity, 0.25% w/w of HCl, 190°C by 3 h). In that study, 84.3% of ethanol yield was achieved after 72 h of fermentation using Pichia stipitis CBS 6054. 4.

Conclusion Glycerol-acid pretreatment was found effective for a considerable hydrolysis yield

even at mild conditions. The influence of different variables was determinate and the parameters of the pretreatment process were optimized. The main compositional modification in the SCB was related to a decrease in hemicellulose content and considerable value of enzymatic digestibility of cellulose fraction was obtained in the optimized pretreatment condition. Potential use of glycerol for pretreatment of lignocellulosic biomass was shown, mainly considering the high glycerol availability due to increase in biodiesel production. Author information Corresponding author [email protected] Conflict of Interest The authors declare that they have no conflict of interest. Acknowledgements The authors gratefully acknowledge the Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica (CONCYTEC/CIENCIACTIVA-Peru, Process number 219-



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2014) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPqBrazil, process number 449609/2014-6).


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Figure captions Figure 1. Solid composition and cellulose hydrolysis yield of SCB pretreated with sulfuric acid diluted in water or 95% of glycerol solution at temperature of 121°C Figure 2. Glucose consumption, ethanol production and cells growth prolife during fermentation of enzymatic hydrolysate by Saccharomyces cerevisiae



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Fig. 1 Solid composition and cellulose hydrolysis yield of SCB pretreated with sulfuric acid diluted in water or 95% of glycerol solution at temperature of 121°C


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Fig. 2 Glucose consumption, ethanol production and cells growth prolife during fermentation of enzymatic hydrolysate by Saccharomyces cerevisiae



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Table 1 Experimental results for 25-1 factorial design: solid composition of pretreated SCB and cellulose hydrolysis yield Experimental variables (coded values in parenthesis) Runs

Composition of pretreated SCB (%)

Cellulose hydrolysis yield (%)

Temperature (°C)

Reaction time (min)

Solid loading (%)

Glycerol concentration (%)

Acid concentration (%)

1

80 (-1)

10 (-1)

2.5 (-1)

85 (-1)

3 (+1)

43.5

24.5

27.7

23.6

2

120 (+1)

10 (-1)

2.5 (-1)

85 (-1)

0.5 (-1)

61.5

10.1

28.2

43.3

3

80 (-1)

60 (+1)

2.5 (-1)

85 (-1)

0.5 (-1)

43.4

24.3

27.5

29.3

4

120 (+1)

60 (+1)

2.5 (-1)

85 (-1)

3 (+1)

68.0

6.0

28.4

61.8

5

80 (-1)

10 (-1)

7.5 (+1)

85 (-1)

0.5 (-1)

38.4

25.6

30.3

24.4

6

120 (+1)

10 (-1)

7.5 (+1)

85 (-1)

3 (+1)

56.9

12.9

30.7

43.8

7 8 9 10 11 12 13 14 15 16 17 18

80 (-1)

60 (+1)

7.5 (+1)

85 (-1)

3 (+1)

120 (+1) 80 (-1) 120 (+1) 80 (-1) 120 (+1) 80 (-1) 120 (+1) 80 (-1) 120 (+1) 100 (0) 100 (0) 100 (0)

60 (+1) 10 (-1) 10 (-1) 60 (+1) 60 (+1) 10 (-1) 10 (-1) 60 (+1) 60 (+1) 35 (0) 35 (0) 35 (0)

7.5 (+1) 2.5 (-1) 2.5 (-1) 2.5 (-1) 2.5 (-1) 7.5 (+1) 7.5 (+1) 7.5 (+1) 7.5 (+1) 5 (0) 5 (0) 5 (0)

85 (-1) 95 (+1) 95 (+1) 95 (+1) 95 (+1) 95 (+1) 95 (+1) 95 (+1) 95 (+1) 90 (0) 90 (0) 90 (0)

0.5 (-1) 0.5 (-1) 3 (+1) 3 (+1) 0.5 (-1) 3 (+1) 0.5 (-1) 0.5 (-1) 3 (+1) 1.75 (0) 1.75 (0) 1.75 (0)

40.2 49.4 54.0 54.0 46.9 61.1 58.3 52.3 42.3 58.5 48.3 47.6 47.1

27.1 19.0 8.2 15.3 14.8 11.3 12.2 20.6 24.9 9.6 16.8 17.3 17.9

27.6 28.4 30.1 31.0 30.5 26.4 24.3 27.9 26.0 33.1 32.3 31.3 30.8

37.8 45.6 24.2 65.6 38.1 52.6 27.8 35.5 24.3 61.9 24.6 23.8 17.9

19


Cellulose

Hemicellulose

Lignin

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Table 2 25-1 factorial design: analysis of effects estimated for response variables composition of pretreated SCB and cellulose enzymatic hydrolysis

Factors Temperature (°C)

Cellulose Std. Effect t p-value Err

Composition of pretreated SCB Hemicellulose Lignin Std. Std. Effect t p-value effect t Err Err

Enzymatic hydrolysis p-value effect

Std. Err

t

p-value

11,83 2,96

3,98

0,001*

-7,10

1,20 13,93

0,000*

1,26

1,26

0,99

0,336

22,57

4,69

4,80 0,000*

Reaction time (min)

-1,14

2,96

-0,38

0,707

0,95

2,62

-2,71

0,017*

-0,28

1,26

-0,22

0,823

7,90

4,69

1,68

0,116

Solid loading (%)

-4,51

2,96

-1,52

0,152

4,67

2,62

0,36

0,722

-0,18

1,26

-0,14

0,884

-4,67

4,69

-0,99

0,338

Glycerol conc. (%)

3,26

2,96

1,09

0,291

-4,07

2,62

1,78

0,097**

0,06

1,26

0,04

0,961

2,55

4,69

0,54

0,596

Acid concentration (%)

2,98

2,96

1,01

0,332

-2.70 2,62 -1,55

0,144

1,06

1,26

0,84

0,415

10,15

4,69

2,16 0,050*

*significant at 95% confidence level, ** significant at 90% confidence level



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Table 3 Solid composition of SCB after pretreatment with glycerol in acid medium (solution with 90% of glycerol and 7.5% of solid loading) and cellulose hydrolysis yield Experimental variables (coded values in parenthesis) Run

Composition of pretreated SCB (%)

Temperature A/B ratio* Reaction Cellulose Hemicellulose (°C) (%) time (min)

Lignin

Cellulose hydrolysis yield (%)

1

120 (-1)

5 (-1)

20 (-1)

39.0

24.6

28.6

10.0

2

130 (+1)

5 (-1)

20 (-1)

37.7

26.0

28.7

19.6

3

120 (-1)

15 (+1)

20 (-1)

41.0

21.5

32.1

30.4

4

130 (+1)

15 (+1)

20 (-1)

54.9

25.0

30.0

51.0

5

120 (-1)

5 (-1)

60 (+1)

49.0

23.2

25.5

32.7

6

130 (+1)

5 (-1)

60 (+1)

50.6

19.1

26.1

39.0

7

120 (-1)

15 (+1)

60 (+1)

46.3

19.6

27.9

32.3

8

130 (+1)

15 (+1)

60 (+1)

46.7

21.4

29.9

64.1

9

116,59(-1.68)

10 (0)

40 (0)

42.5

24.9

27.8

17.1

10

133,41(+1.68)

10 (0)

40 (0)

47.8

17.0

34.6

58.5

11

125 (0)

1,59(-1.68)

40 (0)

43.1

26.0

26.6

19.5

12

125 (0)

18,41(+1.68)

40 (0)

50.6

22.2

30.1

42.6

13

125 (0)

10 (0)

6,36(-1.68)

44.2

26.7

27.8

30.0

14

125 (0)

10 (0)

73,64(+1.68)

51.6

15.4

28.2

43.6

15

125 (0)

10 (0)

40 (0)

44.7

20.0

32.5

46.8

16

125 (0)

10 (0)

40 (0)

46.2

19.9

37.4

43.0

17

125 (0)

10 (0)

40 (0)

47.6

17.0

36.0

40.9

18

125 (0)

10 (0)

40 (0)

46.0

18.5

35.3

36.9

19 125 (0) 10 (0) *Acid/SCB mass ratio (%)

40 (0)

45.4

18.4

36.0

41.6


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Table 4 Analysis of variance (ANOVA) for the adjusted quadratic model for cellulose hydrolysis yield of SCB pretreated by acid-glycerol organosolv method Sum of squares

Source

Degrees of Mean Square freedom

F-value

p-value (Prob> F)

Model

3165.298

5

633.0596

24.1163

< 0.0001

X1

1392.974

1

1392.974

53.06512

< 0.0001

X2

974.2718

1

974.2718

37.11473

< 0.0001

X3

468.3056

1

468.3056

17.84003

0.0010

X22

163.2154

1

163.2154

6.217663

0.0269

X1X2

166.5313

1

166.5313

6.343981

0.0257

Residual

341.2536

13

26.25027

Lack of Fit

289.9616

9

32.21795

2.512513

0.1945

Pure Error

51.292

4

12.823

3506.552

18

Total

Significant*

not significant*

R-square: 0.8653 X1=Temperature (°C), X2=Acid/SCB mass ratio (%), X3= Reaction time (min). *at 95% confidence level



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For Table of Contents Only


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Organosolv Pretreatment of Sugar Cane Bagasse for Bioethanol

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