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Application of optimized alkaline pretreatmentfor enhancing the anaerobic digestion of differentsunflower stalks varietiesFlorian Monlau a , Quentin Aemig a , Abdellatif Barakat a , Jean-Philippe Steyer a & HélèneCarrère aa INRA , UR0050, Laboratoire de Biotechnologie de l'Environnement, Avenue des Etangs,F-11100 , Narbonne , FranceAccepted author version posted online: 28 May 2013.Published online: 20 Jun 2013.

To cite this article: Florian Monlau , Quentin Aemig , Abdellatif Barakat , Jean-Philippe Steyer & Hélène Carrère (2013)Application of optimized alkaline pretreatment for enhancing the anaerobic digestion of different sunflower stalks varieties,Environmental Technology, 34:13-14, 2155-2162, DOI: 10.1080/09593330.2013.808247

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Environmental Technology, 2013Vol. 34, Nos. 13–14, 2155–2162, http://dx.doi.org/10.1080/09593330.2013.808247

Application of optimized alkaline pretreatment for enhancing the anaerobic digestion ofdifferent sunflower stalks varieties

Florian Monlau, Quentin Aemig, Abdellatif Barakat†, Jean-Philippe Steyer and Hélène Carrère∗

INRA, UR0050, Laboratoire de Biotechnologie de l’Environnement, Avenue des Etangs, F-11100 Narbonne, France

(Received 2 February 2013; final version received 15 May 2013)

The use of lignocellulosic residues such as sunflower stalks (SS) for the production of bioenergy such as methane is apromising alternative to fossil fuels. However, their recalcitrant structure justifies the use of pretreatment to enhance theaccessibility of holocelluloses and their further conversion into methane. First, different conditions of alkaline pretreatment(i.e. duration and NaOH concentration (g/100 g TS) at a fixed temperature of 55◦C) were tested to enhance the methanepotential of the stalks of the Serin sunflower (193 mL of methane per gram of volatile solids (VS)). The greatest improvementto the methane potential (262 mL CH4 g−1 VS) was observed at 55◦C, 24 h, 4 g NaOH/100 g TS. Fourier Transform Infraredspectra highlighted an accumulation of lignin in the digestate and the degradation of holocelluloses during the anaerobicprocess, both for pretreated and untreated SS. In a second stage, this optimum condition for alkaline pretreatment (55◦C,24 h, 4 g NaOH/100 g TS) was applied to the stalks of three other varieties of sunflower. Alkaline pretreatment was effectivein the delignification of the stalks of the different sunflower varieties, with lignin reduction varying from 23.3% to 36.3% VS.This reduction of lignin was concomitant with the enhancement of methane potential as compared to that of raw SS, with anincrease ranging from 29% to 44% for the different SS.

Keywords: lignocellulosic material; thermo-alkaline pretreatment; chemical composition; crystallinity; methane potential;infrared spectra

1. IntroductionRecently, the European Union revised its objective ofthe share of renewable energy in total energy consump-tion, raising it to 20%, with a 10% share for renewablefuels in the overall transport fuel supply.[1] The conver-sion into methane of lignocellulosic materials, in particularby-products from agriculture, using anaerobic digestion(AD) technology is one of the promising alternatives tofossil fuels for energy.[2,3] Among such by-products, sun-flower residues, especially the stalks represent an interestingfeedstock for methane production. In 2009, sunflowerswere grown world-wide on 24 million ha.[4] The crop isused to produce oil for the needs of the oil industry andbiodiesel production. Sunflower stalks (SS) are thus presentin huge quantities, have few suitable end uses and are gen-erally burnt in the fields, causing environmental pollution.Methane production from sunflower straw has been inves-tigated by Antonopoulou et al. [5] who found a methanepotential of 240 mL CH4 g−1 sunflower straw.[5]

However, such lignocellulosic substrates as SS presenta major problem for biomethane production on accountof their complex structure which limits their biodegrad-ability.[6] Composed mainly of cellulose, hemicelluloses

∗Corresponding author. Email: carrere@supagro.inra.fr†Current address: INRA, UMR IATE 1208, Ingénierie des Agro polymères et Technologies Emergentes, 2, place Pierre Viala F- 34060Montpellier, France.

and lignin, only the holocelluloses (cellulose and hemi-celluloses) can be converted into methane through ADprocesses.[3,6,7] Recently, some studies have shown thatthe lignin content and, to a lesser extent, the crystalline cel-lulose content have a negative impact on methane produc-tion from lignocellulosic residues.[8,9] Thus, a pretreatmentstep is often required to modify the lignocellulosic struc-ture and obtain a consequent increase in their conversioninto methane. To this end, different pretreatment methods(physical, thermo-chemical and biological or combinationsof them) which were initially developed for bioethanol pro-duction can be applied to enhance the AD of lignocellulosicresidues.[7,10]

Among these various types of pretreatment, alkaline andthermo-alkaline pretreatments that result in the delignifi-cation seem to be a promising way to enhance methanepotential from lignocellulosic residues.[7] By way of exam-ple, pretreatment of miscanthus with 12% NaOH at 70◦Cfor 4 h led to 77% of delignification compared with the rawsubstrate.[11] Moreover, Datta [12] suggested that alkalinepretreatment leads to the saponification of the uronics bondsbetween hemicelluloses and lignin, facilitating the diffu-sion of the hydrolytic enzymes.[12] Consequently, these

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recent years during, alkaline and thermo-alkaline pretreat-ments to enhance methane potentials have been widelyinvestigated on various lignocellulosic substrates such assorghum forage,[13] wheat straw,[13] corn stover,[14,15]grass sillage,[16] rice straw,[17] Cynara stalks [18] andbarley waste.[19] Among alkaline pretreatment applica-tions, Zhu et al. [14] have observed an enhancement of themethane potentials of corn stover of 40% after pretreatmentat 20◦C for 24 h and a concentration of 5% of NaOH.[14] Byapplying thermo-alkaline pretreatment (200◦C, 10 min, 5%NaOH) on rice straw, an increase of the methane potentialof 122% was noticed.[17] Similarly, after alkaline pretreat-ment (160◦C, 20 min, 1.4% NaOH), an increase of 90% ofthe methane potential of Cynara stalks was observed.[18]However, to make the process economically viable, alkalinepretreatment parameters (i.e. temperature, residence timeand chemical concentration) should be optimized regard-ing the methane potential performances. In an earlieststudy, Monlau et al. [20] investigated the impact of vari-ous temperatures (30◦C, 55◦C and 80◦C) on the methanepotential of SS, using a fixed duration and fixed NaOHconcentration.[20] The temperature of 55◦C was found tobe the best for enhancing the methane potential.[20] There-fore, the first objective of this study was to investigatethe parameters of residence time and NaOH concentrationfor one SS variety. Then, the effect of optimized alkalinepretreatment condition on one sunflower stalk variety wasinvestigated for the stalks of three other varieties regardingboth chemical composition and methane potential.

2. Material and methods2.1. Raw materialsSubstrates used were SS of different varieties (NK-Kondi,Naturasol, Serin 1 and Serin 2). First, all SS samples weredried at 37◦C for 48 h and then milled into a particle sizeof 2–3 mm using a cutting mill SM-100. The ‘Serin 1’ and‘Serin 2’ stalks come from the same variety but they werenot grown in the same place and their storage conditionswere different. In fact, the ‘Serin 1’ and ‘Serin 2’ SS werecollected two weeks and two months after harvest, respec-tively. Table 1 summarizes the chemical compositions ofthe four SS varieties.

2.2. Alkaline pretreatmentAlkaline pretreatment was tested on the different varietiesof SS with a solid loading of 35 g TS/L.[20,21] Alka-line pretreatment was carried out in 600 mL flasks agitatedat 150 rpm on an ‘Edmund Butler’ heating shaker seriesSM-30-control. Different durations (3, 6, 12, 24 and 36 h)and NaOH concentrations (0.5, 2, 4, 6 and 10 g/100 g TS)were investigated. After pretreatment, biochemical methanepotentials (BMPs) tests were done on the whole slurry anda part of this slurry was filtered at 0.25 mm to separate theliquid and solid fractions. The solid fractions were dried at60◦C for 24 h.

2.3. BMP assayTreated and untreated samples were digested in batchanaerobic flasks. The volume of each flask was 600 mL,with a working volume of 400 mL, the remaining 200 mLserving as headspace. The inoculum used was granularsludge from a mesophilic anaerobic digester of a sugarfactory. The sludge was composed of 142 ± 1 g TS/Land 118 ± 2 g VS/L. Its initial pH was 7.3. Each flaskcontained: macroelements (NH4Cl, 286 mg L−1; KH2PO4,108 mg L−1; MgCl2, 65 mg L−1; CaCl2, 32 mg L−1), oli-goelements (FeCl2, 20 mg L−1; CoCl2, 5 mg L−1; MnCl2,1 mg L−1; NiCl2, 1 mg L−1; ZnCl2, 0.5 mg L−1; H3BO3,0.5 mg L−1; Na2SeO3, 0.5 mg L−1; CuCl2, 0.4 mg L−1;Na2MoO4, 0.1 mg L−1), a bicarbonate buffer (NaHCO3,2.6 g L−1), an anaerobic sludge at 5 g VS L−1 and the sub-strate, untreated or alkaline pretreated at 5 g TS L−1.[22]Once the flasks were prepared, a degasification step withnitrogen gas was carried out to obtain anaerobic condi-tions. The initial pH value was adjusted to 7 with 37%HCl. The bottles were closed with air-tight red butyl rub-ber septum-type stoppers. Duplicate bottles were incubatedat 35◦C.

2.4. Analytical methodsThe stalks were analysed for total solids (TS) andvolatile solids (VS) in accordance with the APHA stan-dard method.[23] The carbohydrates (glucose, xylose andarabinose) and uronic acids (galacturonic and glucuronic)

Table 1. Composition of four SS varieties. Values correspond to mean ± standard deviation ofmeasurement performed in duplicate.

Parameters Serin 1 Serin 2 Naturasol NK-Kondi

TS (% wet weight) 96.4 (±0) 93.9 (±0.7) 93.9 (±1.3) 93.8 (±0)VS (% wet weight) 89 (±0.4) 78.1 (±2.1) 83.5 (±3.9) 87.4 (±0.7)VS/TS 0.92 0.83 0.89 0.93Uronic acids (g/100 g VS) 2.2 (±0.3) 2.2 (±0.3) 1.3 (±0.3) 7.6 (±0.3)Cellulose (g/100 g VS) 25.1 (±1.7) 20.8 (±0.1) 23.3 (±0.9) 34 (±0.5)Hemicelluloses (g/100 g VS) 11.5 (±1.2) 9.2 (±0.1) 9.4 (±1) 20.8 (±0.8)Lignin (g/100 g VS) 32.5 (±0.6) 25.1 (±1) 33.5 (±2.2) 29.7 (±0.6)

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contents of SS, both untreated and pretreated, weremeasured in duplicate using a strong acid hydrolysis pro-tocol derived from Effland.[24] All SS and solid residuesof alkaline pretreated SS were milled into 1 mm using anIka Werke MF 10 cutting mill. Samples (200 mg) were firsthydrolyzed with 12 M H2SO4 acid for 2 h at room tempera-ture, then diluted to reach a final acid concentration of 1.5 Mand kept at 100◦C for 3 h. The insoluble residue was sepa-rated from the supernatant by filtering on paper fibreglass(GF/F, WHATMAN). This insoluble residue was washedwith 50 mL of deionised water and then placed in a crucible.The crucible and the fibreglass were dried at 100◦C for 24 hto determine the content of Klason lignin. The supernatantwas filtered with nylon filters (0.2 μm). High-pressure liq-uid chromatography analysis was used to quantify thefollowing compounds: glucuronic acid, galacturonic acid,glucose, xylose and arabinose. The analysis was done witha combined Water/Dionex system, using a BioRad HPX-87H column at 50◦C. The solvent was 0.005 M H2SO4 andthe flow-rate 0.3 mL min−1. A Refractive Index detector(Water R410) was used to quantify carbohydrates. The sys-tem was calibrated with glucuronic acid, galacturonic acid,glucose, xylose and arabinose (Sigma-Aldrich). In orderto evaluate the impact of alkaline pretreatment on SS, thecontent of cellulose and hemicelluloses were determinedfrom the monomeric sugars composition. Cellulose is apolymer of glucose and hemicelluloses consist of branchedchains of sugars whose units include mostly pentoses suchas xylose and arabinose. Consequently, the cellulose andhemicelluloses content can be determined as follows:

Cellulose(%TS) = Glucose(% TS)

1.11, (1)

Hemicelluloses (% TS)

= [Xylose(%TS) + Arabinose(%TS)]1.13

, (2)

where 1.11 is the ratio of the molecular weights of glucoseto glucan (180/162) and 1.13 is the ratio of the molecularweights of xylose and arabionose to xylan (150/132).

Biogas volume was monitored by the water displace-ment method. Acidified water (pH = 2) was used to mini-mize the dissolution of carbon dioxide in the water. Biogascomposition was determined using a gas chromatograph(Varian GC-CP4900) equipped with two columns. The firstcolumn (Molsieve 5A PLOT) was used to separate O2,N2, CH4 and CO, and the second (HayeSep A), to sepa-rate CO2 from other gases. The temperatures were 30◦C forthe oven and 100◦C for the injector and the detector. Thedetection of gaseous compounds was done using a thermalconductivity detector. The calibration was carried out witha standard gas composed of 25% CO2, 2% O2, 10% N2 and63% CH4.

2.5. FT-IR assessment and crystallinity calculationFourier Transform Infrared (FT-IR) spectroscopy was usedto visualize the chemical composition and crystallinitychanges induced by AD both with and without alkalinepretreatment. FT-IR spectra were collected in the 4000-600 cm−1 range using a Nexus 5700 spectrometer (Ther-moElectron Corp.) with a built-in diamond attenuated totalreflectance single reflection crystal and with a cooled mer-cury cadmium telluride detector. Spectra were recorded inabsorption mode at 4 cm−1 intervals with 64 scans, at roomtemperature. Four spectra were recorded for each sampleand all spectra pretreatment was analyzed using Omnicv7.3.software. Crystallinity of cellulose for the four SSbefore and after pretreatment and after AD was investigatedby the determination of the Lateral Order Index (LOI) whichis the ratio of the heights of the band at 1430 cm−1 to theband at 898 cm−1.[25,26]

3. Results and discussions3.1. Impact of variations of duration and NaOH

concentration at a fixed temperature (55◦C)First, different pretreatment durations (3, 6, 12, 24 and 36 h)were investigated at a fixed temperature (55◦C, optimaltemperature determinated by the earlier study) and fixedalkaline concentration (4 g NaOH/100 g TS) for ‘Serin 1’SS. For pretreatment durations less than 24 h, i.e. 3, 6and 12 h, no significant increase in methane potential wasrecorded compared to the raw substrate (Figure 1(a)). Onthe contrary, both durations of 24 and 36 h were found effec-tive in enhancing the methane potential as compared toraw sunflower. The highest methane potential was observedafter 24 h with 262 (±12) mL CH4 g−1 VS, correspondingto an increase of 36% as compared to raw SS and wasconsequently selected as the fixed variable for the exper-imental series described below where variations in alkalineconcentration were investigated.

Alkaline concentrations (0.5, 2, 4, 6 and 10 gNaOH/100 g TS) were studied at a fixed temperature (55◦C)and fixed duration (24 h) using Serin 1 SS (Figure 1(b)).From 0.5 g NaOH/100 g TS to 4 g NaOH/100 g TS, anincreasing tendency of the methane potentials was noticed.For higher concentrations than 4 g NaOH/100 g TS, themethane potentials were observed to decrease. Our observa-tions are in disagreement with Xie et al. [16] who noticed agradual methane enhancement by increasing the NaOH con-centration from 1% to 7.5% (g/100 g TS) at 100◦C for 48 h.In our study, the alkaline concentration of 4 g NaOH/100 gTS was found to have the best impact, with an increase ofmethane production from 193 (±16) mL CH4 g−1 VS forraw SS to 262 (±12) mL CH4 g−1 VS.

To sum up, at the fixed temperature of 55◦C, a concen-tration of 4% NaOH and duration of 24 h seem to be theoptimum parameters for enhancing the methane potentialof Serin 1 SS.

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Figure 1. Optimization of alkaline pretreatment parameters: (a)duration and (b) NaOH concentration at fixed temperature (55◦C).

3.2. Monitoring of the AD process by FT-IRmethodology

Changes in the composition and structure of SS after ADwere investigated using FT-IR spectroscopy. In Figure 2,the fingerprints (1700–800 cm−1) of the FT-IR spectra are

Table 2. LOI and H lignin/H carbohydrates ratios for sunflowerstalk ‘Serin 1’, both alkaline pretreated or untreated, before andafter AD (mean and standard deviation of 4 values from 4 spectra).

LOI H lignin/HSamples (H 1430/H 897) carbohydrates

SS 0.91 (±0.08) 0.13 (±0.03)Alkaline pretreated SS 0.89 (±0.06) 0.11 (±0.01)SS digested 1.1 (±0.1) 0.21 (±0.02)Alkaline pretreated SS digested 1.8 (±0.3) 0.25 (±0.02)

represented for sunflower stalks, both untreated and pre-treated (55◦C, 24 h, 4 g NaOH/100 g TS), before and afterthe AD process. The peaks of FT-IR spectra were assignedas follows: 1511 cm−1 is characteristic of aromatic skele-tal vibration C=C of lignin; 1430 cm−1 is assigned to C−Hdeformation in cellulose; 1375 cm−1 is assigned to deforma-tion in cellulose and hemicelluloses; 1160 cm−1 is assignedto C−O−C vibration in holocelluloses; and 898 cm−1 toC−H deformation in cellulose.[27,28]

The most significant difference in spectra between undi-gested and digested SS was observed for the peaks at 898and 1511 cm−1. First, it is interesting to note that the peaks at1511 cm−1, which is related to the lignin content, increasedafter the AD process whereas the peak at 898 cm−1, relatedto the amorphous cellulose, decreased after AD. Thesevariations were quantified by determining the H Lignin/Hcarbohydrates ratio, which is the relative intensity of ligninpeaks at 1511 cm−1 as opposed to the sum of intensity car-bohydrates peaks at 1430, 1375, 1160 and 898 cm−1. Thisratio was calculated after the AD process, both with andwithout alkaline pretreatment on SS, as given in Table 2.

After AD, the H lignin/H carbohydrates ratio was higherthan that of both untreated and pretreated SS, indicating

8001 1001 4001 700

d

c

b

a

Figure 2. Fingerprint region (1700–800 cm−1) of the FT-IR spectra of samples of sunflower stalk ‘Serin 1’: (a) raw and undigested, (b)raw and digested, (c) alkaline pretreated and undigested and (d) alkaline pretreated and digested. Spectra represented are the mean of fourassays for each sample.

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Table 3. Chemical composition of the stalks of four sunflower varieties, untreated and alkaline pretreated. Valuescorrespond to the mean of two replicates of independent values ± standard deviation (error bars). The solubilizationof each compound after alkaline pretreatment is expressed in %.

Biochemical composition (g/100 g initial VS)

Samples % VS initial solubilized Uronic acids Cellulose Hemicelluloses Lignin

Serin 1 2.2 (±0.3) 25.1 (±1.7) 11.5 (±1.2) 32.5 (±0.6)Serin 1 (NaOH) 22.5 0.85 (±0.2) 24.2 (±1.2) 8.8 (±0.4) 24.9 (±1.0)Solubilization (%) 61.3 3.4 23.2 23.3Serin 2 2.2 (±0.3) 20.8 (±0.1) 9.2 (±0.1) 25.1 (±1)Serin 2 (NaOH) 17.8 0.98 (±0.1) 20.4 (±0.5) 8.5 (±0.3) 18.4 (±0.8)Solubilization (%) 55.2 2.0 8.0 26.4Naturasol 1.3 (±0.3) 23.3 (±0.9) 9.4 (±1) 33.5 (±2.2)Naturasol (NaOH) 22.5 1.6 (±0.0) 19.4 (±3.8) 7.8 (±0.5) 22.4 (±0.5)Solubilization (%) 0.0 16.9 16.7 33.0NK-Kondi 7.6 (±0.3) 34 (±0.5) 20.8 (±0.8) 29.7 (±0.6)NK-Kondi (NaOH) 18.4 2.2 (±0.0) 34.3 (±0.0) 15.4 (±0.8) 18.9 (±0.5)Solubilization (%) 71.7 0.0 26.0 36.3

that the lignin content of SS increased compared to the con-tent of holocelluloses, which were degraded by AD. AfterAD, this ratio was slightly higher for alkaline pretreatedSS (0.25 ± 0.02) than for those untreated (0.21 ± 0.02),indicating that alkaline pretreatment improved the degra-dation of holocelluloses. Yang et al. [27] also followed thechemical changes in the lignocellulosic substrate Spartinaalterniflora during AD using FT-IR spectroscopy.[27] Theyinvestigated various ratios of H Lignin/H carbohydrates(H 1511/H 1430, H 1511/H 1375, H 1511/H 1160 and H1511/H 898 cm−1) and all these ratios were twice as greatas that of undigested S. alterniflora. For instance, the ratioH 1511/H 898 increased from 2.52 to 7.56 after AD.[27]

Because the bands at 1430 and 898 cm−1 are sensitive tothe amount of crystalline cellulose and amorphous celluloserespectively,[25,29] the LOI ratio can be used to follow thechanges in cellulose crystallinity. This ratio was calculatedas given in Table 2. First, it is interesting to note that alkalinepretreatment did not seem to act on the cellulose crys-tallinity insofar as the LOI ratio remained almost the samefor pretreated (0.89 ± 0.06) and untreated (0.91 ± 0.08) SS.

Such observations are in accordance with our earlierstudy which investigated the impact of various types ofthermo-alkaline, thermo-oxidative and thermo-acid pre-treatments on cellulose crystallinity.[20] Both alkaline andoxidative pretreatment were found to be ineffective in reduc-ing the content of crystalline cellulose and only 13% of crys-talline cellulose removal was observed after chlorhydricacid pretreatment.[20] Nevertheless, the LOI ratios werehigher after the AD process, with or without alkaline pre-treatment, suggesting that during such a process, amorphouscellulose is degraded to a much greater degree than crys-talline cellulose. Such observations are in agreement withHayashi et al. [30] who suggested that during enzymatichydrolysis of cellulose the readily accessible amorphousregions are more efficiently hydrolyzed, resulting in anaccumulation of crystalline cellulose.[30] The significant

difference of LOI value observed between digested SS(1.1 ± 0.1) and alkaline pretreated SS digested (1.8 ± 0.3)is probably due to the fact that alkaline pretreatmentfavoured the accessibility and degradation of amorphouscellulose during AD.

3.3. Impact of alkaline pretreatment (55◦C, 24 h, 4%NaOH) on the chemical composition of stalks fromdifferent sunflower varieties

The chemical composition (cellulose, hemicelluloses,uronic acids and lignin) of stalks of four sunflower vari-eties, with or without alkaline pretreatments (55◦C, 24 h, 4 gNaOH/100 g TS), was investigated. Results are presentedin % initial VS given in Table 3. For all four untreated SS,the sum of the four compounds (uronic acids, cellulose,hemicelluloses and lignin) accounted for 57.3–92.16% ofthe initial VS, suggesting either that part of the matter wasnot quantified (i.e. lipids, proteins or other sugars) or thatexternal microorganisms or fungi were present in the SSand interfered with the accurate determination of VS.

First, the uronic acids concentration, originating bothfrom acetyl groups of hemicelluloses and from pectin, wasinvestigated. Uronic acids content of 1.3%, 2.2%, 2.2%and 7.6% VS were observed for, respectively, Naturasol,Serin 1, Serin 2 and NK-Kondi stalks. Except for Natura-sol SS, alkaline pretreatment was found effective in uronicacids removal, with reduction greater than 55%. Holocel-luloses content varied between 30% and 37% except forNK-Kondi SS which had a higher holocelluloses contentof 55% VS. Partial hemicelluloses removal varying from8% (Naturasol) to 26% (NK-Kondi) resulted from alkalinepretreatment. For each sunflower variety, low or no cellu-lose removal was observed after alkaline pretreatment. Thisresult can possibly be ascribed to the physical protectionafforded by lignin and hemicelluloses that hinders cellulosedegradation.[31] Finally, lignin contents varied from 25%

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Figure 3. Methane potential (mL CH4. g−1 VS initial) from untreated and alkaline (55◦C, 24 h, 4 g NaOH/100 g TS)-pretreated SS forthe following varieties: (a) Serin 1, (b) Serin 2, (c) NK-Kondi and (d) Naturasol.

VS (Serin 2) to 33.5% VS (Naturasol). A considerable dif-ference in lignin content was found for the same variety,Serin 1 and Serin 2. One explanation may be that Serin 2was left in the field for a longer time and was thus exposedto fungi and microorganisms present in the soil capable ofbreaking down lignin.[32–34] Alkaline pretreatment wasfound effective in removing part of the lignin, the reductionranging from 23.3% (Serin 1) to 36.3% (NK-Kondi). Suchresults are in accordance with several studies in the literaturewhich have highlighted that alkaline pretreatment is effec-tive in delignification.[7,16] For instance, Xie et al. [16]noted that an alkaline pretreatment at 100◦C with a NaOHconcentration up to 1 g/100 g VS resulted in the removalof more than 21% of lignin from grass silage.[16] More-over, such results are in agreement with Zhu et al. [14] whosuggested that alkaline pretreatment is effective in deligni-fication by preserving most of the carbohydrates, cellulosein particular.[14]

3.4. Impact of alkaline pretreatment on methanepotential of the stalks of four sunflower varieties

Batch anaerobic assays were carried out at 35◦C for 70days on the stalks of four varieties of sunflower, with andwithout alkaline pretreatment. Results of methane potentialexpressed in mL CH4 g−1 VS are presented in Figure 3.

Alkaline pretreatment was shown to be effective inimproving methane potential with increases of 29%, 38%,43% and 44% for, respectively, NK-Kondi, Naturasol, Serin1 and Serin 2. Similar results were obereved by Sambusiti

et al. [13] by applying alkaline pretreatment with 10 gNaOH/100 g TS at 40◦C for 24 h with an increase ofmethane potential of 29% and 42% respectively for ensiledsorghum forage and wheat straw.[13] Lehtomaki et al.[35] have also observed an increase of the methane poten-tials 17% after alkaline pretreatment (4 g NaOH/100 g TS,25◦C, 24 h) on grass hay.[35] This increase can be par-tially explained by the decrease in the lignin content ofalkaline pretreated SS as previously suggested by otherstudies that found good correlations between the lignin con-tent and the methane potential.[8,36] However, in our caseonly a poor correlation (R2 = 0.46) was observed betweenthe lignin content and the methane potentials suggestingthat probably other chemical and structural parametersaffect the methane potential. For instance Monlau et al.[9] have found that crystalline cellulose negatively affectsthe methane potentials of lignocellulosic residues also.[9]On the contrary, hemicelluloses, protein and soluble sugarscontents positively affect the methane potentials.[9] More-over, some other parameters such as the accessible surfacearea or the pectin content can probably negatively affectthe methane potentials. Indeed, Pakarinen et al. [37] haverecently shown that the removal of pectin, a polymer ofgalacturonic acids, can significantly increase the hydroly-sis of holocelluloses of various lignocellulosic residues.[37]Moreover, Frigon et al. [38] have shown that the removalof pectins can significantly increase the methane poten-tials of switchgrass. For example, by applying enzymaticpretreatments polygalacturonase at 50 U g−1 VS or pectate-lyase at 6313 U g−1 VS to switchgrass, the rise in the

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methane potential was, respectively, 72% and 42% whencompared to raw switchgrass.[38]

Such results are interesting as they show that alkalinepretreatment can be efficiently applied to enhance signif-icantly the methane potential from the stalks of variousvarieties of sunflower. However, the large range of methanepotential increase varying between 29% and 44% sug-gests that probably an optimization step should be realizedfor each SS variety or directly on a mix of SS varietiesaccording their availability for AD. Moreover, it should beinteresting in future work to extend these optimized con-ditions to other lignocellulosic substrates that present verydifferent chemical and structural parameters from SS as theefficiency of one type of pretreatment certainly depends onthe nature of the lignocellulosic substrates as suggested byWyman et al.[39]

4. ConclusionsThe alkaline pretreatment of SS was best at 55◦C, 24 h,4% NaOH. This pretreatment led to the increase, rangingfrom 29% to 44%, of the methane potentials of the stalkssampled from four varieties of sunflower. Delignificationvarying from 23.3% to 36.3% VS observed during suchpretreatment can be one explanation for the improvement inmethane potential. FT-IR spectroscopy showed that duringthe AD process, holocelluloses of SS are degraded, leadingto an accumulation of lignin in the digestate. Moreover, ofthe different types of cellulose, amorphous cellulose wasdegraded to a greater degree during the AD process.

AcknowledgementsThe authors are grateful to ADEME, the French Environment andEnergy Management Agency, for financial support in the form ofF. Monlau’s PhD grant.

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