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Innovative deconstruction of biomass induced by dry chemo-mechanical activation: Impact on enzymatic hydrolysis and energy efficiency Charlotte Loustau-Cazalet, Cecilia Sambusiti, Patrice Buche, Abderrahim Solhy, Florian Monlau, and Abdellatif Barakat ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00194 • Publication Date (Web): 14 Mar 2016 Downloaded from http://pubs.acs.org on March 30, 2016

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ACS Sustainable Chemistry & Engineering

Innovative deconstruction of biomass induced by dry chemo-mechanical activation: Impact on enzymatic hydrolysis and energy efficiency C. Loustau-Cazalet,† C. Sambusiti,† P. Buche,† A. Solhy,*,# F Monlau,† E. Bilal,†† M. Larzek,# and A. Barakat*,† †

IATE, CIRAD, Montpellier SupAgro, INRA, Université de Montpelier, 34060, Montpellier, France. E-mail: [email protected]

#

Center for Advanced Materials, Mohammed VI Polytechnic University. Lot 660 - Hay Moulay Rachid. 43150 Ben Guerir, Morocco. E-mail: [email protected]

††

R&D OCP, OCP Group, Complexe industriel Jorf Lasfar. BP 118 El Jadida, Morocco.

Keywords: Biomass, Sugars, Chemo-mechanical, Activation, Energy

ACS Paragon Plus Environment

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ABSTRACT Lignocellulose fractionation to chemicals and biofuels often requires severe conditions and consume high energy. Although some of these pretreatment processes are nowadays developed at pilot scale, are not always cost effectives and they are responsible of environmental impacts. Recently, mechanocatalysis pretreatment emerged as promising technologies for the activation and deconstruction of lignocellulosic biomass. In particular, these dry processes have the potential to limit the use of solvent and the production of liquid effluents. In this study we propose an innovative one-pot eco-friendly approach based on the combination of dry chemical and vibro-ball-milling (VBM) for biomass activation, coupled to enzymatic conversion without external source of heating and without assistance of any organic solvent. NaOH activation coupling to VBM fractionation for 10, 30 and 60 min was compared to H3PO4, H2O2, betaine and betaine-Cl activation. NaOH-VBM-10 min appears more effective in sugars production compared to other chemical-mechanical activation. NaOH-VBM of 10 min consumed less energy and resulting in higher energy efficiency compared to others chemical-mechanical activation. Therefore, NaOH-VBM-10min appears the most suitable and interesting pretreatment for the production of sugars and biofuels from corn stover biomass.


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INTRODUCTION The bioconversion of lignocellulosic biomass has been extensively studied in the past 30 years. In spite of such research endeavors, the effect of enzymatic and chemical pretreatments on lignocellulose transformation is still poorly understood because of competing effects including physical properties of substrate, synergies between enzymatic and chemical catalysts and mass transfer. The structural heterogeneity and complexity of lignocellulosic matrix constituents, such as crystallinity of cellulose microfibrils, accessible surface area, porosity and matrix polymers are responsible of the recalcitrance of lignocellulosic materials.1-3 Biomass pretreatment is consequently an essential step in order to reduce cellulose crystallinity, increase accessible surface area and porosity and separating the major constituents of biomass (cellulose, hemicellulose, lignin, phenolic acids, ash, etc.) from which a very rich chemistry can be then developed. The objective of pretreatments depends on the process type and biomass structure. For instance, treatment technologies, aimed to produce biofuels and chemicals, target changes in lignocellulosic matrix properties, thus rendering carbohydrates and lignin more accessible to enzymatic hydrolysis.4-6 Pretreatments methods are classified into different categories: mechanical, chemical, physicochemical and biological or various combinations of them. Lignocellulose fractionation often requires severe conditions (high temperature and high pressure, strong acid and alkaline concentrations…).2,7-9 Although some of these pretreatment processes are nowadays developed at pilot scale, are not always cost effectives and they are responsible of environmental impacts, hampering theirs large-scale commercialization. Furthermore, in some cases, pretreatments not only generate liquid effluents but also modify and convert carbohydrates and lignin into fermentation inhibitors by-products, necessary to be treated or co-valorize. All these downstream steps dramatically impact the final cost of extraction


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process. Recently, dry chemo-mechanical treatments or mechanocatalysis emerged as promising technologies for the activation and deconstruction of lignocellulosic biomass.10-14 In particular, these dry processes have the potential to limit the use of solvent, the production of liquid effluents and avoid the separation of solid/liquid fractions.1,2,11,15 This paper proposes an innovative and ecofriendly approach based on the combination of dry chemical and vibro-ballmilling (VBM) for biomass deconstruction, coupled to enzymatic hydrolysis. This one-pot dry chemo-mechanical activation and fractionation approach allows carbohydrates to be separated from lignin, making them more accessible to enzymes without external source of heating and without assistance of any organic solvent (i.e. ethanol, ionic liquid, choline...) compared to previous studies.10-12

Scheme 1. Solvent and effluent free deconstruction of corn stover (CS) induced by dry chemomechanical treatment.

A schematic representation of the process developed in this study is provided in scheme 1. This work is distinguished from previous studies for: (1) organic-free and thermal-free process


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(2) a process that concomitantly dissociates and separates lignin and carbohydrates from biomass (3) a process that uses a low water content (solid/liquid ratio: 5/1). To the best of our knowledge, the dry chemo-mechanical realized at high solid loading and organic solvent-free without external heating source coupled to enzymatic hydrolysis has never been explored for the dry activation and deconstruction of biomass. Thus the final aim to this study is to demonstrate the potentiality of this technology within the framework of biorefinery.

RESULTS AND DISCUSSION In this study, corn stover (CS) was used as model lignocellulosic substrate. CS was obtained from a farm located in the south of France (Languedoc-Roussillon region). According to a typical procedure, sample was coarsely cut to less than 2 mm by knife milling prior to chemical impregnation (solid/liquid ratio: 5/1) directly in the ball mill with a spray system at ambient temperature (Scheme 1). The impregnate CS was then ultrafine milled using a vibratory-ball milling (VBM), equipped with an energy measurement system, at ambient temperature with a frequency of 15 s-1 by varying the VBM time (10, 30 and 60 min). The different fractions were then analyzed in terms of particle size, chemical composition and physical structure. Total energy requirement was also measured. Then, fractions were enzymatically hydrolyzed.


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Control

NaOH

H2O2

Betaine

H2O2 Urea

Betaine Cl

H3PO4

60 55 50

Par cle Size (µm)

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45 40 35 30 NaOH

25 20 15 10 0

10

20

30

40

50

60

Vibro-Ball Milling (min) Figure 1. Particle size (µm) of corn stover (CS) in function to milling time (min) for different dry chemo-mechanical treatments.

As shown in Fig.1, NaOH-VBM was more effective in the reduction of particle size compared to the other chemo-mechanical treatments: the combination NaOH-VBM-treated CS resulted in fine particles with an average diameter (D50) of 27.3 µm (10 min), 18.5 µm (30 min) and 34.1 µm (60 min) compared to 50.5 µm (10 min), 28.0 µm (30 min) and 23.2 µm (60 min) for VBMtreated CS (control). The increase in particle size of NaOH-VBM for 60 min was likely due to an aggregation of the ultrafine particles produced, caused probably by a deconstruction of lignocellulosic matrix under chemical and compression mechanical stresses that produced reactive particles. On the other hand, no significantly effect was observed by coupling H2O2, betaine, H2O2-urea and betaine-Cl to VBM compared to control; resulting in average particle diameter sizes (D50)


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of about 44-49 µm; 25-29 µm and 22-25 µm for 10, 30 and 60 min of VBM, respectively (Fig.1). This decreasing of particle size with increasing of VBM time can be related to no aggregation of ultrafine particles compared to powder resulting from NaOH-VBM. It interesting to note that NaOH-VBM is more effective in the reduction of particles size, compared to other alkaline and acidic co-treatments. It has been demonstrated that particle size reduction enhances the production of glucose or reducing sugars.1,11,16 Dasari and Berson reported an increase of about 55% of the glucose production during enzymatic hydrolysis of cellulose after particle size reduction from 590 µm to 33 µm.17 Moreover, Zeng et al. demonstrated that corn stover particles of 53-75 µm are 30% more susceptible to hydrolysis than 425-710 µm corn stover particles.18 Lignin, cellulose, hemicelluloses, and ash were determined for different CS-treated fractions. As shown in Fig. 2, the composition of the control (without chemical treatment) is very different to that of the chemical treated fractions. Moreover, the composition of NaOH-CS fraction is different compared to that of other chemical treated fractions. In particular, it is richest in cellulose (57.4%) and ash, and poorest in lignin. This difference in chemical composition between the NaOH treated CS-fraction and the other chemical treated fractions supports clearly the potentiality offered by NaOH for lignin solubilisation. The solubility of mechanical and chemo-mechanical treated CS was evaluated and compared with cellulase-xylanase cocktails (Fig. 4A) and with cellulase (Fig. 4B). The solubility of carbohydrates and lignin in presence of enzymes and without enzymes was determined using the equation 1: weight of residue after hydrolysis Eq 1 Solubility % w/w = 100 × weight of sample before hydrolysis


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Figure 2. Biochemical composition of corn stover (CS) in function to different dry chemomechanical treatments.

Phenolic acids play also a very important role in the cohesion of lignocellulosic polymers. The quantity of phenolic acid monomers (ferulic (FA), p-coumaric (p-CA), syringic (SA) and vanillic (VA) acids) and dimers (di-FA) was determined (Fig. 3). It can be seen in Fig. 3 that the control fraction (VBM) fraction was also richer in phenolic acids such as in p-coumaric acid (pCA), ferulic acids, vanillic acid (VA) and ferulic acid dimer (di-FA) compared to NaOH-VBM treated-CS. It has been previously reported that grassy plants contain ferulic bridges between lignin and carbohydrates via ester-linked ferulic and p-coumaric acids forming lignincarbohydrate complexes (LCC), which give the recalcitrant complex structure of the lignocellulosic matrix.19,20


8

’-d iFA 8, 5

VA 8 -d ,5’b iFA en zo fu ra n

iFA ’-d

A

O4

)-S

8-

(E

A 8, 5’ -d iF A

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(Z )-S

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Figure 3. Phenolic acid monomers and dimers for VMB (control) and NaOH-VBM.

Cellulose crystallinity (CrI) of mechanically and dry chemo-mechanically treated CS was determined only for the NaOH-VBM treated-CS compared to VBM treated-CS. Results indicate that no significantly effects were observed between VBM and NaOH-VBM treatment for 10, 30 and 60 min on cellulose crystallinity. Without chemical treatment the CrI decreased from 60.5% for untreated CS (0 min of VBM) to 57.5, 56.8 and 57.3% for 10, 30 and 60 min of VBM. As shown in Fig. 4, dry chemo-mechanical treatments generally increased CS solubility compared to VBM treated-CS (control). NaOH-VBM was more effective in the solubilization of carbohydrates-lignin materials compared to other treatments. NaOH-VBM solubilized approximately 62% of CS in buffer without enzymes (Fig.4C) compared to about 90% with


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cellulase (Fig.4B) and more than 94% in the presence of cellulose and xylanase cocktails (Fig.4A). No effect of milling time was observed with NaOH. On the other hand, VBM-treated CS coupled to H2O2, betaine, H2O2-urea and betaine-Cl resulted in a solubilization in buffer without enzyme (Fig. 4C) of about 30% to 50% from 10min to 60min of VBM, respectively. In contrast, about 50 to 70% were solubilized in the presence of enzymes from 10 to 60min of VBM respectively, which depends on the milling time. Generally, NaOH is efficient in lignin solubilization and can be successfully used to break ether and ester bonds in lignin/phenoliccarbohydrates complexes.


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Control

NaOH

H2O2

Betaine

H2O2 Urea

Betaine Cl

H3PO4

100

A Solubility (% w/w DM)

90 80 70 60 50 40 30 100

Solubility (% w/w DM)

B 90 80 70 60 50 40 100

C 90

Solubility (% w/w DM))

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 70 60 50 40 30 0

10

20

30

40

50

60

Vibro-Ball Milling (min)

Figure 4. Solubility of corn stover (CS) for various dry chemo-mechanical treatments (A) with cellulose + xylanase cocktails, (B) with cellulase enzyme and (C) in buffer without enzyme. However, carbohydrates and lignin solubilization after NaOH- VBM deconstruction are higher compared to others pretreatments. In addition, carbohydrates and lignin solubilization increased in presence of enzymes, which allows degrading cellulose and hemicelluloses polymers into soluble oligomeric and monomeric sugars.


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Results of enzymatic conversion of mechanically and dry chemo-mechanically treated-CS samples (Fig. 5) showed that glucose yields of chemo-mechanically treated-CS were greater than those of control and that dry NaOH-VBM treatment was more effective in enzymatic accessibility and bioconversion of carbohydrates: NaOH > H2O2 > H2O2-urea ≈ H3PO4 ≈ betaine ≈ betaine-Cl. With respect to VBM, glucose yields increased with the milling time. In contrast, glucose yields remained constant after 10, 30 and 60 min of VBM-NaOH in presence of cellulase and xylanase cocktails (Fig. 5A). Glucose yield mg/g Cellulose in Biomass

1100

A

NaOH

Control

NaOH

1000 900 800 700 600 500 400

H2O2

Betaine

20

30

H2O2 Urea

Betaine Cl

H3PO4

50

60

1100

Glucose yield mg/g Cellulose in Biomass

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B 1000 900 NaOH

800 700 600 500 400 0

10

40

Vibro-Ball Milling (min)

Figure 5. Glucose yields of different dry chemo-mechanical treatments of corn stover (CS) with (A) cellulase-xylanase cocktails and (B) only with cellulase cocktail.


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Results suggest that, to obtain the best glucose yield, NaOH-VBM is preferred and more effective. However, the corresponding milling time has to be chosen accordingly to total energy demand or requirement (ETER). The positive results in terms of solubility and enzymatic conversion enhancement obtained by the NaOH treatment were mainly due to a substantial removal of lignin and cleaving of lignin-carbohydrates complex (LCC), higher than that occurred during the other treatments and not due to a reduction of cellulose crystallinity. Therefore, it is not surprising that the control was lower accessible to enzymes and produced lower sugars compared to NaOH-VBM. Fig. 6 shows the consumption of glucose and the production of ethanol during the fermentation of VBM (control) and NaOH-VBM pretreated-CS. Results revealed that the yeast quickly consumed glucose after inoculation at 2-10 h and the totality of ethanol was produced during the first 24 h in all fermentations. As expected the bioethanol production trends were in accordance with results obtained during enzymatic hydrolysis for VBM and NaOH coupling to VBM of pretreated-CS and whatever the milling time. In particular, a significant effect of mechanical fractionation on bioethanol production was noticed. Results also show that VBM-60 min gave the best result in terms of ethanol yield (112 g ethanol/g DM), compared to 10 min and 30 min of VBM. Among pretreatments, whatever the fractionation method, similar ethanol yields (up to 221 g ethanol/g DM) were obtained after NaOH pretreatment, which gave the best results compared to control (Fig 6).


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Figure 6. Ethanol and glucose yield obtained during fermentation of corn stover treated with (A) NaOH coupling to VBM-10, -30 and -60 min compared to (B) control without chemical treatment.

The total energy requirement (Eq.2) and energy efficiency (Eq.3) was also used to compare the performance of VBM.11,16,21 The amount of glucose recovered after enzymatic hydrolysis was divided by the total energy requirement during VBM (Eq 3).

E!"#

&

$ P& dt = ' Eq 2 m


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ɳ=

Glucose kg Eq 3 ETER kWh

The ETER in ball milling process depend strongly on time (Hideno et al. 2009). VBM fractionation at 60, 30 and 10 min with and without NaOH were compared in term of energy requirement and energy efficiency. The ETER of VBM treated-CS decreased significantly from 8.57 kWh.kg-1 to 4.92 and 1.60 kWh.kg-1 for 60, 30 and 10 min of VBM, respectively (Table 1). Therefore NaOH-VBM consumed lower energy compared to VBM without chemical treatment. It can be seen in Table 1, an ETER of 8.37 kWh.kg-1, 4.32 and 1.42 were obtained for 60, 30 and 10 min of VBM with NaOH treatment coupling to VBM fractionation, respectively.

Table 1: Total energy requirement and energy efficiency ETER (kWh.kg-1 DM

Glucose (g.kg-1 DM)

ɳ (kg.kWh-1)

VBM-10 min

1.60

207.4

0.130

VBM-30 min

4.92

226.5

0.046

VBM-60 min

8.57

258.7

0.030

NaOH-VBM-10 min

1.42

396.4

0.279

NaOH-VBM-30 min

4.32

406.4

0.094

NaOH-VBM-60 min

8.37

391.5

0.047

Fractionation Process

ɳ: Energy efficiency, DM: Dry matter

It can also be seen (Table 1) that the maximum glucose yield produced was 406.4 g kg-1DM of CS after NaOH-VBM-30 min and enzymatic hydrolysis, whereas only a maximum of 258.7 g.kg-1 DM was obtained after VBM-60 min without chemical co-treatment. Therefore, NaOHVBM-60min and VBM-60min consumed the highest amount of energy (ETER). As a result,


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NaOH-VBM-60min and VBM-60min showed the lowest ɳ, being 0.048 and 0.030 kWh.kg-1, respectively compared to 0.279 and 0.130 kg glucose kWh-1 obtained for NaOH-VBM-10min and VBM-10min, respectively. Table 1 showed also that after NaOH-VBM-10min the total energy consumption was the lowest with an excellent sugar recovery. In another study, Zhu and Pan reported that steam explosion and disk milling consumed the highest amount of energy.12 As a result, steam explosion and disk milling without co-treatment have the lowest ɳ (0.63 and 0.088 kg glucose kWh-1, respectively). According to the study of Mathew et al., the maximum sugar production after acid pretreatment and hydrolysis was obtained from biomass pre-treated for 90 min.22 However, the ɳ was found to be lower when biomass was pre-treated for 90 min than for 60 min. Hence, it can be concluded that the highest ɳ obtained was 0.94 kg glucose kWh-1 from a pretreatment time of 60 min. According to Hideno et al., in terms of energy efficiency a WDM pretreatment of one operation cycle was preferred to 10 cycles.11 They compared also the efficiency energy of ball milling (BM) and WDM. In BM, the monomeric sugar yields after enzymatic hydrolysis increased with milling time. They reported that BM treatment at 60 min resulted in lower ɳ compared to DM-5 min and -10 min for the pretreatment of rice straw. The highest ɳ obtained was 0.078 kg glucose kWh-1, for rice straw after BM at 5 min. Table 2 reported a comparative study of various pretreatment technologies developed for corn stover saccharification. Normally, energy efficiency (kg glucose extracted/kWh of total energy requirement) is used to compare the pretreatment performances but literature data are scares (Table 2). However, glucose yields, solvents and chemical catalysts amount were used to compare the pretreatments. En-factor, which is defined as the ratio of the mass of waste per unit


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of product was also calculated (Eq 3). A high En-factor means more waste and consequently greater negative environmental impact23: 12 − factor =

Total waste kg Eq 4 Glucose reducing sugars kg

Total amount of reactant (kg) = biomass + chemical catalyst + water. Total waste = Total amount of reactant (kg) – glucose or total reducing sugars (Kg)

The highest glucose recovery from corn stover (about 380 mg.g-1) was obtained after steam explosion at 3.5 MPa during 90 min,24 but resulting in a high En-factor of 19.9 due to high solid/liquid ratio (1/7). On the other hand, steam pretreatment produced about 80% of glucose with an En-factor of 70.0.25 AFEX26 and hot compressed water pretreatments27 of corn stover produced about 90% of glucose and resulting in an En-factor of about 7.0 and 36.7, respectively. This difference, due to the quantity of water used in the pretreatment, was 15 times higher for AFEX pretreatment. This study was generally fine from the green chemistry standpoint, with a low En-factor of 0.9-1.4 (Table 2).

According to a close comparison of discussed protocols and methodologies reported on corn stover, NaOH-VBM treatment appeared the simplest and the less consuming technology which can effectively improve the rate of saccharification and bioethanol production without solvent and heating requirement, and with an En- factor of approximately 0.9. This would mean minimizing waste generation while maximizing value of the lignocellulosic feedstock.

Table 2: Comparison of various corn stover (CS) pretreatment with the technologies developed in this study


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Ref.

Pretreatment conditions AFEX: Ammonia loading is 1:1 gNH3/g dry biomass and water loading is 0,6:1 gH2O/g dry biomass with residence time of 8h at 40°C

[26]

Steam pretreatment: Impregnation for 30 min with aqueous solution where the mass fraction was 1% acetic acid and the ratio of liquid to solids is 20:1 by weight. After press, the steam pretreatment is at 200°C at 10 min in pressure of 3MPa Steam explosion: residence time at 90 min with pressure at 3,5 Mpa and concentration at 146 g/L of dry material Dilute sulfuric acid pretreatment: 2% w/v sulfuric acid; 60 min; 135°C Cutting milling: 414 µm Hot compressed water: 10% w/v of the total working volume with 180240°C Hot compressed water: 10% w/v of the total working volume with 180240°C and 5mmol/L ammonium sulfate

[25]

[24]

[28]

[27]

Twin screw extrusion with NaOH solution: 99°C and 325 rpm, impregnation time 10h

[29]

Planetary ball milling with water: ball speed of 350 rpm, solid liquid ratio of 1:10, raw material particle size with 0,5 mm and number of balls of 20 and grinding for 30 min

[30]

This study

Vibratory ball milling (VBM): raw particle size 2 mm, 10 min, 15 Htz VBM coupled to alkaline treatment with NaOH: particle size 2 mm, 10% NaOH (w/w), soli/liquid ratio of 5:1, 10 min of VBM at 15 s-1

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Glucose yield (% w/w)

Water or solvent (L.kg-1 biomass)

Chemical catalyst (kg.kg-1 biomass)

En-factor

72 h of hydrolysis at 1% glucan loading, 50°C, and rotated at 200 rpm, Spezyme Cp (15 FPU/g cellulose) and Novozyme 188 (30 CBU/g cellulose)

90

0.6

1

7.0

96h of hydrolysis at 5% w/v, 40°C with agitation, Cellic C-Tec 2 at 7,5 FPU/g

80

20

0.2

70.0

98

7

0

19.9

73

10

0.2

35.1

10

0

0

28.1

82

10

0

38.1

90

10

0.7

36.7

82

2

0.06

10.9

88

10

0

29.7

56

0

0

1.4

98

0.1

0.2

1.0

Enzymatic hydrolysis conditions

72h at 50°C in the acetate buffer with a Accelerase 1500 at 30 FPU/g substrate solid loading was 5% w/v, 72h at 50°C and 150 rpm, with Ctec 2 at 60 FPU/g 72h at 50°C and 180 rpm, Cellulase at 15 FPU/g and beta glucosidase at 65 CBU/g Solid loading 2%, 50°C for 48 h, 20 FPU/substrate of cellulase and 5 IU/substrate of betaglucosidase; Solid loading 6%, 130 rpm at 50°C in sodium citrate buffer at 50 mM with cellulase at 15 FPU/g glucan and xylanase at 200 IU/g Solid loading 5 % (w/v), Cellulase and Xylanase at 20 FPU/g biomass, 72h, 37°C, 100 rpm

CONCLUSIONS Results indicate clearly that the coupling of chemical and mechanical deconstruction of biomass within an eco-conception (economy of atom and energy and without effluent) is a promising technology of biomass fractionation and valorization into chemicals and biofuels. In particular, results show that vibro ball-milling (VBM) fractionation coupled to NaOH treatment was more effective in the reduction of particles size and in sugars production compared to other


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chemical-mechanical treatments. VBM-NaOH at 10 min consumed less energy, resulting in higher energy efficiency than VBM and VBM-NaOH at 30 and 60 min. Therefore, NaOH-VBM10min appears the most suitable and interesting pretreatment for the production of sugars from corn stover biomass.

EXPERIMENTAL SECTION Dry chemo-mechanical treatments. Sodium hydroxide (NaOH), hydrogen peroxide (H2O2) betaine, H2O2-urea and betaine-Cl were dissolved in distilled water to adjust the alkaline concentration at 10 g of catalyst/100g of CS. The alkaline solutions were made with the amount of water required to adjust the moisture content of 100 g of corn stover (CS) to 20 % (dry basis) equivalent to a biomass/liquid ratio of 5/1 and a high material concentration of 5 kg.L-1. The chemical treated CS samples were equilibrated for 5h at room temperature (25°C). Then, untreated and chemical pretreated CS samples (1-4 mm) were subjected to a vibratory-ball milling (VBM, Retsch, MM 400, Germany). For this purpose, 2 g of sample was added to a 20 mL milling cup containing 1 stainless steel ball (diameter 2 cm) and then milled for 10, 30 and 60 min at room temperature.

Physical and chemical characterization. After VBM, the particle size of control and chemical treated CS were analyzed by a laser granulometry (Mastersizer 2000, Malvern Instrument, France). The total energy requirement of CS milling was measured using a wattmeter. The power active, active electric energy (Wh), frequency hertz and time were logged into a computer card at 1-s intervals. Phenolic acids were also analyzed according to Antoine et al. [18]. Ester-linked phenolic acids were saponified under Argon (oxygen-free) at 35 °C in 2N NaOH. An internal standard (2,3,5 trimethoxy-(E)-cinnamic acid (TMCA), T-4002, Sigma Chemical Co., St Louis, USA) was added before adjusting pH to 2. Phenolic acids were then extracted with diethylether and quantified by high performance liquid chromatography (HPLC). The Crystallinity index (CrI) was determined using X-ray-diffraction (XRD). Powder XRD patterns were


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recorded on a Bruker diffractometer D8 Advance. The measurements were conducted on powder compacted on small mats. DRX data were collected from 2θ = 5° to 50° with a step interval of 0.02°. The degree of crystallinity can be expressed as the percentage crystallinity index (% CrI).

Enzymatic hydrolysis and fermentation. Enzymatic hydrolysis of CS samples was performed using an enzyme cocktail (Trichoderma Longibrachiatum C9748) obtained from Sigma Aldrich (20 FPU g-1 biomass). Enzymatic hydrolysis was conducted at a solid concentration of 5 % (w/v) in 50 mM sodium acetate buffer (pH 5.0) at 37 °C for 72 h under stirring. Tests were performed in triplicate. SSF experiments were carried out in 2 mL serum bottles, each containing 7.5% (w/v) dry fraction in potassium phthalate buffer (50 mM, pH5.5), 0.1 mL (20U/g) of Trichoderma Longibrachiatum C9748 enzymes and 0.9 mL of nutrients containing: 9 g L-1 yeast extract, 5 g L-1 urea, 0.5 g L-1 MgSO4·7H2O and 1 g L1

KH2PO4. Flasks were closed and incubated at 37°C for 72 hrs. The experiment was performed in

triplicate.

AUTHOR INFORMATION Corresponding Authors * Abdellatif Barakat: IATE, CIRAD, INRA, SupAgro, Université de Montpellier, 34060, Montpellier, France E-mail: [email protected]

* Abderrahim Solhy: Center for Advanced Materials, Mohammed VI Polytechnic University. Lot 660 - Hay Moulay Rachid. 43150 Ben Guerir, Morocco. E-mail: [email protected] Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT The financial assistance of the INRA towards this research is hereby acknowledged. This work was also supported by a grant from the Office Chérifien des Phosphates in the Moroccan Kingdom (OCP Group). We would like to dedicate this paper to our dear friend and colleague: Dr. Varma Rajender from Sustainable Technology Division, National Risk Management Research Laboratory, U.S. Environmental Protection Agency, who has been involved for years in the development of scientific and technological research, and who help us as a young research team for overwhelm. Again, we want to thank Dr. Varma Rajender for his enthusiasm and efficiency in facilitating the valuation of our results of research.

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Innovative deconstruction of biomass induced by dry chemo-mechanical activation: Impact on enzymatic hydrolysis and energy efficiency C. Loustau-Cazalet,† C. Sambusiti,† P. Buche,† A. Solhy,*,# F Monlau,† E. Bilal††, M. Larzek,# and A. Barakat*,†

An innovative strategy to fractionate Lignocellulosic biomass in order to produce biofuel by coupling dry mechanochemical process, and enzymatic hydrolysis, was developed.


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Innovative Deconstruction of Biomass Induced by ... - ACS Publications

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