Ethanol production from halophyte Juncus maritimus using freezing and thawing biomass pretreatment

Renewable Energy xxx (2015) 1e5

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Review

Ethanol production from halophyte Juncus maritimus using freezing and thawing biomass pretreatment Neila Smichi a, Yosra Messaoudi a, Nizar Moujahed b, Mohamed Gargouri a, * a Biocatalysis and Industrial Enzymes Group, Laboratory of Microbial, Ecology and Technology, National Institute of Applied Sciences and Technology, Carthage University, BP 676, 1080 Tunis Cedex, Tunisia b Laboratory of Animal and Food Resources, National Agronomic Institute of Tunisia, Carthage University, 43 Av. Ch. Nicolle, 1082 BelvedereTunis, Tunisia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 October 2014 Received in revised form 5 April 2015 Accepted 5 July 2015 Available online xxx

Juncus maritimus contains (41.5 ± 0.3)% cellulose and (31.34 ± 0.2)% hemicellulose on dry solid (DS) basis and has the potential to serve as a low cost feedstock for ethanol production. Dilute acid or freezing/ thawing pretreatments and enzymatic saccharification were evaluated for conversion of halophyte plant from J. maritimus cellulose and hemicelluloses to monomeric sugars. The maximum concentration of released glucose from J. maritimus (53.78 ± 3.24) g L
1) by Freezing/thawing pretreatment and enzymatic saccharification (55  C, pH 5.0 and 48 h) using CellicCTec2 from Novozymes and (49.14 ± 5.24) g L
1 obtained by dilute acid pretreatment. The maximum yield of ethanol from acid pretreated enzyme saccharified J. maritimus hydrolyzate by Saccharomyces cerevisiae strain was (84.28 ± 5.11)% of the theoretical yield with a productivity of (0.88 ± 0.16)g L
1 h
1. It was (90.87 ± 1.94)% of the theoretical yield with a productivity of (1.04 ± 0.10) g L
1h
1 for freezing/thawing pretreated plant and enzymatic hydrolysis by CellicCTec2. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Juncus maritimus Dilute acid pretreatment Freezing/thawing pretreatment Enzymatic saccarification Alcoholic fermentation

1. Introduction Renewable energies are gaining more interest as fossil energy becomes more scarce and expensive. In fact, the exploitation of different biomass sources may compensate a major part of future energy demands. In this context, lignocellulosic biomass represents a potential candidate for bioethanol production due to its availability and environmental benefits [1]. Bioethanol is mainly produced from food sources such as sugarcane, rice, cassava, and corn. Consequently, the exploitation of a large amount of food sources for bioethanol production would increase food cost [2]. Therefore, researchers are focusing on finding renewable resources other than human food resources. In this context, lignocellulosic materials such as agricultural residues, forestry wastes and municipal materials constitute a potential source for bioenergy production [3]. Lignocellulosic biomass mainly consists of three polymers which are cellulose, hemicelluloses and lignin [4]. Among these components, carbohydrates

* Corresponding author. E-mail addresses: [email protected] (N. Smichi), [email protected] (Y. Messaoudi), [email protected] (N. Moujahed), [email protected] (M. Gargouri).

(cellulose and hemicelluloses) can be saccharified and eventually fermented in order to obtain bioethanol [5]. The pretreatment process is the most important step in cellulose-to-ethanol technology, since it can solubilize hemicelluloses, reduce cellulose's crystallinity and increase the materials' porosity [4]. There is a number of available pretreatment technologies including physical, biological, physical-chemical (liquid hot water, steam explosion ammonia fiber explosion, Instant Controlled Pressure Drop [6,7]) and chemical (acid, alkaline, wet oxidation, ozonolysis) [8]. However, the dilute acid is the most widely used method for lignocellulosic material pre-treatment due to its relatively low cost, ease of use [9] and high efficiency [10]. It removes and hydrolyzes up to 90% of hemicelluloses making the cellulose fraction more accessible to enzymatic hydrolysis. While, this pretreatment presents important drawbacks related to the formation of toxic compounds (furfural and hydroxymethyl furfural), issued from sugar degradation, as inhibitors for the fermentation step. Also, the mineral acids are equipment stainlesscorrosive. In addition, the environmental problems caused by the waste streams liberated from the dilute acid pretreatment process [10]. Its mechanism comprises series of reactions. First, the protons are diffused through the wet lignocellulosic matrix followed by the protonation of the ether-oxygen link between the monomeric

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Please cite this article in press as: N. Smichi, et al., Ethanol production from halophyte Juncus maritimus using freezing and thawing biomass pretreatment, Renewable Energy (2015), http://dx.doi.org/10.1016/j.renene.2015.07.010

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N. Smichi et al. / Renewable Energy xxx (2015) 1e5

sugars. Then, the rupture of the ether bond was achieved for generation of a carbocation. Finally, the latter undergoes a solvation with water in order to generate sugars monomers, oligomers and polymers depending on the ether connection that is broken with regeneration of proton [11]. Glycophytes represent a large proportion of plant species, which are sensitive to high soil salinity in contrast to halophytes that naturally tolerate such conditions. In fact, halophytes can be found in alkaline semi-deserts, salt marshes, steppes, and sea coasts. The use of halophytes plants as a bioenergy biomass is beneficial since they don't compete with conventional crops for high quality soil and may be irrigated with saline water [12,13]. Consequently, there is a necessity for exploiting saline lands in producing nonfoodligno-cellulosic biomass, which may be later transformed into ethanol without compromising human food production. Hereby, halophytes, which produce plenty of biomass in saline environment, constitute a potential candidate for bioenergy production. In this work, our main objective was to produce ethanol from halophyte biomass using Juncus maritimus as a raw material for bioenergy production. In order to achieve this objective, three steps processes were performed. First, the plant was treated by two various methods, which were dilute acid and freezing/thawing. To our knowledge, the last method has never been used for biomass pretreatment in order to produce ethanol. However, it is used as a conservation process (cryoconservation) in several domains such as food industry [14], medical technology, pharmaceutical industry (i.e. to store drug substances and protein for a long periods) [15], biochemistry and microbiology (i.e. to conserve proteins, tissues and cells) [16]. Several studies indicate that cells' membrane systems are the primary site of freezing injuries in plants [17,18]. In fact, under a temperature around
20  C (frozen state), the plasma membrane of plant cells undergoes a rupture by the formation of a large amount of intracellular ice. When cells are exposed to high temperature (thawed state), the intracellular frozen cells are subjected to further ultra structural changes during the thawing process resulting in serious cell damage [19]. Thus, the freezing/thawing method could be used to breakdown cell walls to enhance the accessibility of their polysaccharides to the hydrolyzing enzymes. Then, an enzymatic saccharification was performed in order to produce fermentable sugar. Finally, the latter was converted into bioethanol using S. cerevisiae yeast.

enzymes that prove effective on a wide variety of pretreated lignocellulosic materials for the conversion of the carbohydrates in these materials to simple sugars prior the fermentation. The endoglucanase activity is standardized on the basis of its activity on carboxymethylcellulose (CMC). A CMC activity unit liberates 1 mmol of reducing sugars in 1 min under specific assay conditions of 50  C and pH 5. The b-glucosidase activity is standardized on the basis of activity on pNPglucoside. One pNPG unit denotes 1 mmole of Nitrophenol liberated from para-nitrophenyl-b-D-glucopyranoside per minute at of 55  C and pH 5. A strain of yeast S. cerevisiae was selected as a model strain for fermentation of simple sugars. The fermentation was achieved using fresh commercial baker's yeast S. cerevisiae (Tunisian Society of yeasts, S. cerevisiae purchased from local market) [20]. 2.2. Dilute acid pretreatment of J. maritimus Milled J. maritimus was slurries in1% H2SO4 (5% on dry solid basis, DM, w/v) and pretreated in an autoclave at 121  C for1 h. The wet material was filtered. The solid residue was washed 3 to 4 times by distilled water to neutralize pH. It was dried at 50  C for 48 h [21]. 2.3. Freezing/thawing pretreatment 5 g of milled J. maritimus were frozen in a conventional freezer (Classical Freezer, LG, GN-392, Tunisia) for 24 h at
20  C [22] and immediately thawing in water bath at 100  C for 15 min just for obtaining a thawed material. The sample was then filtered and dried at 50  C for 48 h. Finally, the residue was reserved for the enzymatic hydrolysis step. 2.4. Enzymatic saccharification The enzymatic saccharification of the diluted acid and the freezing/thawing pretreated J. maritimus was performed for 48 h at 55  C and pH5 adjusted with 5 mM sodium acetate buffer and adding enzymes at each enzyme dose of 1 mg g
1 DS (dry substrate) of J. maritimus [23]. Samples (1 mL) were kept at 20  C before HPLC analysis. These experiments were replicated three times. 2.5. Yeast cultivation

2. Materials and methods 2.1. Materials J. maritimus Lamk. is a salt marsh plant. Plant-shaped tufts grow up to 1 m height. The rhizoma generates long and parallel stems that are all radical, naked, and hard spines at the end. 5-year old J. maritimus was harvested in its native salty ecosystem from the Tunisian region Soliman sabkha (N 36 42 14, E 10 27 21). This region is located on the Mediterranean coast 40 km of Tunisian and characterized by upper semi aridbioclimatic. Average of annual rainfall estimated about 450 mm. Aerial parts were collected in March 2013 corresponding to the flowering period. It was dried at 50  C for 48 h and milled in a hammer mill to attain a particle size of 3 mm. The milled J. maritimus was then stored at room temperature. CellicCTec2 (0,43 pNPG U ml
1, 61, 25 CMC U ml
1) and CellicHTec2 (0,25 pNPG U ml
1, 92, 3 CMC U ml
1) enzymes from Novozymes (Denmark) and Accelerase 1500 (0,25 pNPG U ml
1,38,3 CMC U ml
1) from Genoncor were used for the enzymatic hydrolysis of J. maritimus. There are states of the art on

S. cerevisiae was used as an ethanol fermentation strain. The yeast strain was maintained on agar plates made from 5 g L
1 yeast extract, 5 g L
1 peptone, 20 g L
1 d-glucose, and 20 g L
1 agar. Inoculation flasks were prepared by autoclaving 100 mL of 50 g L
1 glucose, 1 g L
1 KH2PO4, 1 g L
1 MgSO4 þ 7H2O, 5 g L
1 peptone and 5 g L
1 yeast extract. The medium was incubated for 24 h at 30  C with shaking (150 rpm) prior to use. 2.6. Fermentation The batch fermentation experiments were carried out in 100 mL flasks under anaerobic conditions with working volumes of 20 mL. The hydrolysates (15 mL) of the solid fraction resulting of J. maritimus pretreatment were used as substrates with 2 mL of YPX10 (200 g L
1 yeast extract and 400 g L
1 peptone)and 2 mL of the yeast suspension. Fermentation was incubated for 24 h at 37  C with shaking (100 rpm) [24]. These experiments were replicated three times. For calculation of ethanol yield the following equation was applied [25,26].

Please cite this article in press as: N. Smichi, et al., Ethanol production from halophyte Juncus maritimus using freezing and thawing biomass pretreatment, Renewable Energy (2015), http://dx.doi.org/10.1016/j.renene.2015.07.010

N. Smichi et al. / Renewable Energy xxx (2015) 1e5

Ethanol Yield ð%Þ ¼

Measured Ethanol in sample ðgÞ Theoretical Ethanol

Theoretical ethanol (g) ¼ Amount of initial sugar content (g) in fermentation solution X 0.5

Ethanol Productivity PE ¼

Measured Ethanol in sample ðgÞ reactionnel volume ðLÞ  time ðhÞ

2.7. Analytical procedures Neutral detergent fiber (NDF), acid detergent fiber (ADF), cellulose and lignin content were determined by van Soest method. Hemicellulose content of J. maritimus was determined as NDF minus ADF [27]. Ethanol and glucose were chromatographed on an HPLC system equipped with a refractive index detector (Agilent 1200 Series). Before injection, samples were filtered through a 0.20 mm filter. Aliquots of the filtered sample (20 mL) were injected into the HPLC system. The glucose and ethanol were eluted using 1 mM H2SO4 as the mobile phase. The PRONTOSIL120-5-C18-AQ 5.0 mm column (250 mm  4.0 mm) was used at room temperature with a flow rate of 0.3 mL min
1. A complete analysis of the glucose and ethanol was carried out during 25 min. 3. Results and discussion 3.1. Composition of J. maritimus J. maritimus, used in this investigation, contained (41.5 ± 0.3)% cellulose and (31.34 ± 0.2)% hemicelluloses which make up the total carbohydrate content of 72.84% on dry solid basis and 5.7% lignin (Table 1). This composition is comparable to sugarcane which contained 40e43% glucan, 20e24% xylan and 24e27% lignin [28e30], to sorghum which mainly contained 45% of glucan, 28% of xylan, and 22% of lignin [31] and to Sweet sorghum bagasse (41.3% glucan, 17.9% xylan, and 18.2% lignin) [32]. It is also comparable to Corn stover which contains (37.5 ± 0.1)% glucan, (21.7 ± 0.2)% xylan, and (19.3 ± 0.2)% lignin on dry basis [33,34]. Juncus maritimus which produces plenty of biomass, with high level of carbohydrates, using saline resources could be a promising source for bio-energy (bioethanol) production without compromising human food sources.

3

enzymatic hydrolysis was determined by calculating the concentration of the glucosereleasedfrom the cellulose and hemicelluloses conversion. Comparing the two pretreatments, we noted that with the freezing/thawing one resulted in a higher digestibility (susceptibility to hydrolysis) of the pretreated J. maritimus to CellicCTec2, CellicH Tec2 and Accelerase1500 than the one obtained with the 1% sulfuric acid pretreated J. maritimus. The concentration of released glucose (Fig. 1) from the freezing/thawing treated J. maritimus were (53.78 ± 3.24) g L
1, (30.96 ± 0.44) g L
1 and (24.54 ± 1.31) g L
1 with CellicC Tec2, CellicHTec2 and Accelerase 1500, respectively. Even with dilute acid pretreatment, J. maritimus glucose concentration was higher than what was described for corn Stover's. According to the data reported in literature, the glucose concentrations obtained from corn stover were18.3 g L
1 and 36.1 g L
1 from acid and alkaline pretreatments [35], respectively. Consequently, halophyte plant as J. maritimus is potential alternative resource for fuels production. These results can be explained by the fact that the dilute acid pretreatment at severe conditions leads to sugars degradation. The acids, H2SO4 [36,37], HCl [38], HF [39] or CH3COOH [40]used as catalysts, solubilize the hemicellulosic fraction of lignocellulosic matrix. In fact, the pretreatment consists on the releasing of proton that break the heterocyclic ether bonds between momoneric sugars of hemicelluloses and cellulose in order to release xylose, glucose and arabinose and other molecules (furfural and acetic acid) which are considered as unwanted products [41,42]. Aliphatic acids, Furfural, HMF and phenolic compounds are formed from the degradation of sugars and lignin [43]. These compoundscan inhibit enzymatic hydrolysis by at least 50% [44]. These facts combined lead to a decrease in glucose yield. According to the data reported by Noaa and al [45], the dilute acid hydrolysate of poplar biomass contains 0.71 g/100 g of furfural, 1.56 71 g/100 g of acetic acid, 2.41 71 g/100 g of formic acid and 0.04 g/100 g of HMF obtained when Poplar biomass was pretreated in 0.98% (w/v) dilute acid at 140  C for 40 min. It seems that freezing/thawing pretreatment was a very promising pretreatment method for converting raw feedstock to a substrate susceptible to hydrolytic enzymes. The freezing/thawing method can damage cells' membrane system due to the severe dehydration appearing after freezing. Multiple forms of membrane damage can occur due to freeze induced cellular dehydration including expansion-induced-lysis, lamellar-to hexagonal-II phase transitions and fracture jump lesions [46,47].A high concentration of glucose was reached after enzymatic saccharification of sulfuric acid or freezing/thawing

3.2. Enzymatic saccharification of pretreated halophyte plant from J. maritimus 60

Table 1 Composition of Juncus maritimus on dry solid basis.

50

Glucose (g L )

The solid fraction resulting from the 1% dilute acid pretreatment at 60 minor freezing/thawing pretreatment of J. maritimus was subjected to enzymatic saccharification using three commercial enzymatic preparations for 48 h (Cellic C Tec2, Cellic H Tec2 and Accelerase 1500)at 50  C and pH5. The performance of the

40 30

Acid pretreatment freezing/thawing pretreatment

20 10

Component

Dry solids (%,w/w)

Cellulose Hemicellulose Lignin Acid detergent fiber Neutral detergent fiber

41.5 ± 0.3 31.34 ± 0.2 5.7 ± 0.2 47.2 78.54

0 Cellic C-Tec2

Cellic H-Tec2

Accelerase 1500

Enzymes ( mg g DB)

Fig. 1. Released glucose concentration (g L
1) of freezing/thawing or dilute acid pretreatments after enzymatic hydrolysis of Juncus maritimus using Cellic C-Tec2, Cellic HTec2 and Accelerase 1500 enzymes at 50  C for 48 h.

Please cite this article in press as: N. Smichi, et al., Ethanol production from halophyte Juncus maritimus using freezing and thawing biomass pretreatment, Renewable Energy (2015), http://dx.doi.org/10.1016/j.renene.2015.07.010

N. Smichi et al. / Renewable Energy xxx (2015) 1e5

treated J. maritimus with CellicCTec2, these concentrations (Fig. 1) were around (49.14 ± 5.24) g L
1 and (53.78 ± 3.24) g L
1, respectively. The glucose concentration was increased with CellicCTec2. This result can be explained by the efficiency of this enzyme and its composition. In fact, Cellic C Tec 2 enzyme converts cellulose and hemicelluloses, containing polymeric forms of sugars, into hydrolyzed fermentable monomers [48].

60 50 Ethanol yield (%)

4

3.3. Fermentation of J. maritimus hydrolyzates

30

Acid pretreatment freezing/thawing pretreatment

20 10

30

0 Cellic C-Tec2

Cellic H-Tec2 Accelerase 1500 Enzymes (mg g-1 DB)

Fig. 3. Maximum ethanol yield (%) from freezing/thawing or dilute acid treated Juncus maritimus hydrolysates by Saccharomyces cerevisiae.

1.20 1.00 ProducƟvity ( g L h )

The hydrolyzate obtained from freezing/thawing treated J. maritimus, contains (53.78 ± 3.24) g L
1 glucose when fermented with Saccharomyces cerevisae. The ethanol production (Fig. 2) reached (24.95 ± 2.38) g L
1 with (90.87 ± 1.94)% of theoretical ethanol yield (based on the theoretical yield of 0.51 g ethanol g
1 glucose) equivalent to (0.46 ± 0.19) g g
1 and a productivity of (1.04 ± 0.10) g L
1 h
1 after 24 h (Figs. 3 and 4). The fermentation of the hydrolyzates from dilute acid treated J. maritimus by Cellic C Tec2, produced (21.18 ± 3.79) g L
1 of bioethanol (Fig. 2) with a (84.28 ± 5.11)% of theoretical ethanol yield equivalent to (0.42 ± 0.51) g g
1 and (0.88 ± 0.16) g L
1 h
1 of productivity (Figs. 3 and 4). These results can be explained by the presence of some inhibitors in dilute acid hydrolysate inhibiting the alcoholic fermentation. Gia-Luen and al. [21], used similar pretreatment conditions (2% of sulfuric acid, 121  C and 60 min) to produce ethanol from bagasse, silvergrass and rice straw as raw materials. Authors showed that biomass treatment under these conditions released about 90% and 80% of acetic acid from bagasse and rice straw respectively, resulting in a decrease of ethanol yield by inhibiting the fermenting microorganisms. Acetic acid is generated by the hydrolysis of acetyl groups on hemicelluloses. It is noteworthy that bagasse contain 3.4% (w/w) of acetyl group. Another important inhibitor, furfural, was found to be generated by conversion from xylose under the used pretreatment conditions. This study demonstrated that both acetic acid and furfural inhibit the growth of xylose-fermenting microorganisms and thus reducing the ethanol production. While, the ethanol yield was influenced by the presence of toxic compounds such as furfural, hydroxylmethyl furfural and acetic acid formed during the pretreatment of lignocellulosic that inhibit the enzymatic hydrolysis and the fermentation steps. In order to minimize their negative effects on fermentation and to improve its yield, several strategies were developed: i) different physical chemical and biological methods were proposed to detoxify the hydrolysate before the fermentation step by converting the toxic compounds into products that not interfere with metabolism, ii) development of fermentative species that are tolerate the inhibitors

Ethanol cocentraƟon (g L )

40

0.80 0.60

Acid pretreatment freezing/thawing pretreatment

0.40 0.20 0.00 Cellic C-Tec2

Cellic H-Tec2

Accelerase1500

Enzymes (mg g DB)

Fig. 4. The volumetric ethanol productivity (g ethanol L
1 h
1) from conversion of freezing/thawing or dilute acid treated Juncus maritimus hydrolyzates by Saccharomyces cerevisiae.

exciting in the hydrolysate [49]. The ethanol yields obtained in this study were even higher than the previous results (0.40 g g
1) from sorghum [50] and near to 0.48 g g
1 from corncob [51]. According to the data reported in literature, ethanol yields from total fermentable sugars using a C6fermenting strain (S. cerevisiae) reached 89.8% of theoretical ethanol yield and 91.8% for bagasse and straw hydrolysates, respectively, which is in accordance with our results [52]. The results show that halophytes plant from J. maritimus is a potential feedstock for bioenergy. Halophyte plant, abundant in nature, can compete favorably with other classical biomasses for biofuel production that's why it was considerate as suitable lignocellulosic material for its transformation into ethanol.

4. Conclusion

25 20 15

Acid pretreatment freezing/thawing pretreatment

10 5 0 Cellic C-Tec2

Cellic H-Tec2

Accelerase 1500

Enzymes mg g DB

Fig. 2. Ethanol concentration (g L
1) of dilute acid or freezing/thawing treated Juncus maritimus by Saccharomyces cerevisiae.

This study, report, the time in our knowledge, the successful production of cellulosic ethanol from halophyte plant as Juncus maritimus. Enzymatic saccharification, with cellicCtec2, of J. maritimus with dilute acid or freezing/thawing showed a high glucose concentration of (49.14 ± 5.24) g L
1 and (53.78 ± 3.24) g L
1, respectively. The maximum theoretical ethanol yield (90.87 ± 1.94)% was obtained with freezing/thawing treated J. maritimus. Treatment with freezing/thawing could be an efficient alternative for pretreatment of lignocellulosic biomass before enzymatic hydrolysis due to high efficiency. In fact, the pre-treatment can affect the cellulosic region differently and hence facilitates the enzyme treatment and thus the

Please cite this article in press as: N. Smichi, et al., Ethanol production from halophyte Juncus maritimus using freezing and thawing biomass pretreatment, Renewable Energy (2015), http://dx.doi.org/10.1016/j.renene.2015.07.010

N. Smichi et al. / Renewable Energy xxx (2015) 1e5

fermentation to produce the ethanol. In this study, we proposed a new promising pretreatment method (freezing/thawing) of lignocellulosic biomass used to breakdown the cell walls in order to enhance the accessibility of their polysaccharides to enzymatic attack. The advantages of freezing/thawing pretreatment include the absence of chemicals and dangerous reagents and mild environmental conditions (no toxic products and no wastes are released). However, it requires further equipment and a longer processing time. These results confirm also that the halophyte plant J. maritimus could serve as a novel material for the production of ethanol.

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Please cite this article in press as: N. Smichi, et al., Ethanol production from halophyte Juncus maritimus using freezing and thawing biomass pretreatment, Renewable Energy (2015), http://dx.doi.org/10.1016/j.renene.2015.07.010