ORIGINAL ARTICLE

Production of ethanol from the hemicellulosic fractionof sunflower meal biomass

Danielle Camargo & Luciane Sene

Received: 20 June 2013 /Revised: 28 August 2013 /Accepted: 29 August 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Researches on second-generation ethanol, pro-duced from agroindustrial wastes, have demanded specialattention as a possible solution to energy sustainability. Suchproduction is based on lignocellulosic fiber conversion, whichgenerates fermentable sugars that are biotransformed intoethanol. This work aimed at evaluating ethanol productionby the yeast Pichia stipitis ATCC 58376 in the hemicellulosichydrolysate of sunflower meal biomass, a subproduct gener-ated by sunflower oil manufactures. Sunflower meal wassubmitted to dilute acid hydrolysis with 6 % (w /v ) H2SO4 inautoclave, at 121 °C, for 20 min and resulted in ahemicellulosic hydrolysate with high concentration of sugars(24.98 g/L xylose, 26.55 g/L glucose, and 6.51 g/L arabinose)and low amounts of toxic compounds (3.04 g/L total phenols,0.58 g/L acetic acid, 0.40 g/L furfural, and 0.09 g/Lhydroxymethylfurfural). The fermentations of the detoxifiedhydrolysate were conducted in Erlenmeyer flasks at 30 °C,initial pH 5.5, under different agitation speeds (100, 150, and200 rpm). The best ethanol production (8.8 g/L ethanol,yield of 0.23 g/g, and productivity of 0.12 g/L h) wasattained at 200 rpm. The results demonstrate that sun-flower meal is a promising biomass for ethanol productionfrom its hemicellulosic fraction. In addition, the hemicellulosichydrolysate has the advantage of not requiring a sugar concen-tration step, which contributes to the economic viability of theprocess.

Keywords Sunflowermeal . Pentose fermentation . Ethanol .

Pichia stipitis

1 Introduction

Production of renewable fuels, especially lignocellulosic eth-anol, has considerable potential to meet the current demandfor energy as well as to mitigate emissions of greenhousegases to a sustainable environment [1]. This procedure isbased on the utilization of lignocellulosic biomass, such aswood waste and agricultural waste, considered as the bestpotential raw material for producing ethanol, since it is themost abundant source of sugars and does not compete with thefood source [2].

A number of different strategies have been envisioned toconvert the polysaccharides into fermentable sugars. One ofthem consists in the pretreatment of biomass with dilute acids.The resulting hemicellulosic hydrolysate is a liquid rich inxylose, glucose, and arabinose. The cellulosic fraction is solidand can be readily hydrolyzed to glucose with enzymes. Boththe hemicellulosic hydrolysate and the cellulosic hydrolysatecan be fermented to produce ethanol [3].

One of the challenges to commercializing lignocellulosicethanol is the utilization of pentose sugars [4] and is dependenton a robust organism to use the different sugars present inbiomass, including the pentose sugars L-arabinose and D-xy-lose [5, 6]. Among the xylose-fermenting yeasts, Pichia stipitishas shown promising results in the conversion of xylose toethanol from lignocellulosic biomass hydrolysates [7–11].Although several strains of P. stipitis have been studied, thereare few reports on the strain ATCC 59376, and none of themrefer to its performance in hemicellulosic hydrolysates.

Sunflower (Helianthus annuus) is one of the most impor-tant oil seed crops cultivated worldwide for oil extraction,which represents up to 80 % of its economic value [12, 13].Oil cakes/meals are by-products obtained after oil extractionfrom the seeds [14]. After removal of the proteinaceous frac-tion, the remaining lignocellulosic fraction has a high potentialfor use as fermentation source [13].

D. Camargo (*) : L. SenePost-Graduate Program in Agricultural Engineering—PGEAGRI,Center of Exact and Technological Sciences, Western Paraná StateUniversity, Rua Universitária, 2069, Cascavel-PR, CEP85819-110 Cascavel, Paraná, Brazile-mail: daniellecamargodc@hotmail.com

Biomass Conv. Bioref.DOI 10.1007/s13399-013-0096-0

There are many reports in the literature on the use sunflowermeal in diets [15–17]; however, sunflower biomass has scarcelybeen studied as a renewable and cheap resource for biologicalprocesses. Major studies in literature deal with ethanol produc-tion from sunflower seed hulls by P. stipitis NRRL Y-7124[18–20], ethanol by enzymatic saccharification of sunflowerhusks and fermentation by Saccharomyces cerevisiae [21], andconversion of raw sunflower hulls into fermentable sugars bydilute acid pretreatment and enzymatic hydrolysis [22].

The aim of this study was to investigate the feasibility ofsunflower meal hemicellulosic hydrolysate for ethanol pro-duction by P. stipitis ATCC 58376. The influence of agitationspeed on the fermentative performance of this strain was alsoinvestigated.

2 Experimental

2.1 Raw material and dilute acid hydrolysis

Sunflower meal biomass, a by-product generated by oil pro-cessing manufactures, was obtained from CaramuruAlimentos, Itumbiara-GO, Brazil. It was ground to 50 meshand dried at 50 °C for 12 h, and its lignocellulosic compositionwas determined [23]. The biomass was hydrolyzed with 6 %(w /v ) H2SO4 with a biomass/solution ratio of 1:10 w /v, inautoclave, at 121 °C, for 20 min. This condition was definedin preliminary studies (data not shown).

2.2 Hydrolysate detoxification

The hemicellulosic hydrolysate was filtered and detoxified byadjusting the initial pH to 7.0 with Ca(OH)2 and then to 5.5with H3PO4, followed by the adsorption with activated carbonpowder (1 g of charcoal to 40 g of hydrolysate, in a shaker at200 rpm, 60 °C, for 1 h).

2.3 Microorganisms and growth conditions

P. stipitis ATCC 58376 was used in the fermentation experi-ments. Active cultures for inoculation were prepared by growingthe organism in YMP medium (3 g/L malt extract, 3 g/L yeastextract, 5 g/L peptone, and 10 g/L glucose) on a rotary shaker at150 rpm, 30 °C for 24 h. Subsequently, the cells were recoveredby centrifugation (2,000 rpm, 20 min), washed twice with sterilewater, and after new centrifugation, resuspended and inoculatedin the fermentationmedium to an initial concentration of 0.5 g/L,calculated with a standard curve (dry cell mass vs. O.D.600 nm).

2.4 Fermentation tests

The fermentation tests were carried out in 250-mLErlenmeyerflasks, containing 50 mL of the hydrolysate autoclaved at

115 °C for 15 min, and supplemented with 3 g/L malt extract,3 g/L yeast extract, and 5 g/L peptone. The initial pH wasadjusted to 5.5, and the tests were run without pH control. Theflasks were incubated on a rotary shaker at 30 °C, at 100, 150,and 200 rpm for 72 h. Aliquots of 2 mL of each Erlenmeyerflask were withdrawn periodically to determine cell mass, pH,sugars, inhibitors, and production of ethanol and xylitol.

2.5 Analytical methods

The concentrations of glucose, xylose, acetic acid, glycerol,xylitol, and ethanol in the hydrolysate and fermentation me-dium were determined by high-performance liquid chroma-tography (Shimadzu, model 20A), IR detector, columnPhenomenex Rezex ROA Organic Acid-H+ (8 %), 150×7.8 mm, eluent H2SO4 0.005 mol/L, flow rate 0.6 mL/min,at 65 °C. For furfural and hydroxymethylfurfural (HMF)determination, samples were analyzed by liquid chromatogra-phy using the following conditions: column C18 [2] 5um 90A3.9×300 mm; Waters 2487 UV/vis detector at 276 nm; eluentacetonitrile/water (1:8) with 1 % acetic acid; and flow rate0.6 mL/min, at room temperature. The concentrations of allcompounds above were determined from standard curvesobtained with chemicals of high purity (99 % Sigma).

The recovery of pentoses was calculated from the percent-age of separation of pentosans as the mass (xylose plus arab-inose) after acid hydrolysis multiplied by the conversion fac-tor (0.88) over the total mass of hemicellulose obtained fromsunflower meal.

The concentration of total phenols was determined in aspectrophotometer at 760 nm according to the Folin–Ciocalteu method described by Singleton et al. [24] usingvanillin (2-methoxyphenol) (99 %, Synth) as standard.

3 Results and discussion

Sunflower meal biomass utilized in this study presented thefollowing chemical composition: 32.93 % cellulose, 30.90 %hemicellulose, 26.62 % lignin, 5.05 % ash, 27.93 % protein,and 1.60 % lipids. The results reported in the literature for thelignocellulosic composition of sunflower hulls are differentfrom each other and vary between 34 and 53 % cellulose, 17and 35 % hemicellulose, and 11 and 25 % lignin [21, 22, 25].These differences may be partially attributed to differences invariety, climate, and water availability [26]. Besides that, thehigh lignin values obtained in the present work can be possi-bly attributed to the high percentage of protein (27.93 %),which may have been retained in the filter paper during thequantification of insoluble lignin.

The chemical composition of the original hydrolysateobtained after dilute acid treatment of sunflower meal andthe detoxified hydrolysate is shown in Table 1. The condition

Biomass Conv. Bioref.

of hydrolysis used (6 % w /v H2SO4, 20 min) was defined inpreliminary studies (data not shown). This pretreatment waseffective for the solubilization of the hemicellulose, since theremaining solid fraction contained 0.97 % hemicellulose,34.7 % cellulose, and 37 % insoluble lignin. Although xylosewas present in higher concentration in the hemicellulosichydrolysate (26.55 g/L), a high glucose concentration(24.98 g/L) was also observed, which may have originatedfrom some heteropolymers of the hemicellulosic fraction,from the cellulose fraction, and from the starch. Glucoseconcentrations from 26 to 33.5 g/L were found in the diluteacid prehydrolysates of rice hulls (2 % H2SO4, 122 °C, for 20,40, or 60 min) probably because of hydrolysis of starch of thegrain that remains in the hulls [27].

Compounds considered inhibitory to microbial metabolismsuch as furans, phenolics, and acetic acid were also present inthe sunflower meal hemicellulosic hydrolysate. However, thevalues verified did not exceed those usually observed in othertypes of hemicellulosic hydrolysates. In sugarcane bagassehydrolysate obtained under a milder condition (2 %H2SO4, 60 min), similar values of furfural (0.36 g/L)and HMF (0.07 g/L), while a higher concentration ofacetic acid (2.7 g/L) and lower concentration of phenols(0.24 g/L), were observed [27].

The detoxification process utilized in this work resulted ina loss of glucose (17%), xylose (9%), and arabinose (21 %)and a partial removal of phenols (30 %) and acetic acid(77.58 %) compared to the original composition.Hydroxymethylfurfural and furfural were not detected inthe hydrolysate after the detoxification process. Experimentalresults with P. stipitis DSM 3651 showed that furfural caused adelay in sugar consumption rates with increasing concentration,and HMF did not exert a significant effect. However, there wasa synergistic effect due to the presence of acetic acid, furfural,and HMF [28].

Figure 1 shows the cell growth, the consumption of glucoseand xylose, and the production of ethanol by P. stipitis underdifferent agitation speeds (100, 150, and 200 rpm). During thefirst 24 h, a slow growth for all the tested agitations wasobserved, indicating the phase of adaptation of the yeast tothe culture medium. After this period, cell growth was fasterand was seen to be influenced by the increase of agitation. Thehighest cell concentration (4.91 g/L) was attained at 200 rpm.Similar behavior was observed in the ethanol production by P.stipitis NRRL Y-7124 [29] and P. stipitis CBS 6054 [9] inwhich cell growth increased due to the increase of the oxygentransfer rate.

The increase of agitation favored the use of glucose, sinceat 100 rpm this sugar was consumed in 48 h, while at 150 and200 rpm, it was totally assimilated after 36 and 24 h, respec-tively. With P. stipitis NRRLY-7124, glucose was consumedin 12 h of fermentation with high volumetric oxygen transfercoefficients (kLa>3.2 h−1), but when the lowest kLa (0.7 h−1)was used, glucose consumption was extended for 24 h [29].

Although repression of xylose assimilation by glucose waspreviously observed in P. stipitis ATCC 58376 and CBS 6054[30, 31], preferential use of glucose with respect to xylose didnot occur in this work. Xylose assimilation was also affectedby the agitation speed, since at 100 and 150 rpm, a consump-tion of 75 and 72 %, respectively, was observed after 48 h,while at 200 rpm, about 75 % of xylose was consumed in ashorter period of 36 h. With respect to arabinose, it was foundthat the concentration was maintained near 5 g/L (data notshown) in the three evaluated fermentation conditions.

Ethanol production was observed in all runs, and ethanolconcentration increased with the increase of agitation. Thehighest concentration (8.8 g/L) was obtained at 200 rpm andseemed to be correlated to the increase of cell mass. In P. stipitisCBS 6054, oxygen was necessary for ethanol production notmainly because of the redox imbalance, but rather was requiredfor cell growth, the function of mitochondria, and to generateenergy for the transport of xylose [32]. The present results accordwith those observed in the fermentation of the hemicellulosichydrolysate of sugar maple wood by P. stipitis NRRLY-7124, inwhich a correlation between the increase of ethanol concentra-tions and agitation speed (7.70, 7.40, and 14.3 g/L at 150, 200,and 250 rpm, respectively) was reported [33].

The degree of aeration has to be at a certain threshold level,when insufficient aeration leads to very small biomass andethanol formation. An oxygen supply stimulates significantgrowth and ethanol formation depending on aeration rate.Moreover, excessive levels of aeration reduce the ethanolyield because of either product oxidation or cell growth [18].

In this work, the increase of ethanol production rate oc-curred after the exhaustion of glucose in all agitations ana-lyzed, indicating that the yeast efficiently assimilated xyloseto produce ethanol. However, P. stipitis ATCC 58376 had adifferent behavior in synthetic medium with mixed sugars,

Table 1 Concentration of monomeric sugars and inhibitory compoundsin sunflower meal hemicellulosic hydrolysate before and afterdetoxification

Compound Concentration (g/L)

Before detoxificationa After detoxificationa

Glucose 24.98 20.66

Xylose 26.55 24.09

Arabinose 6.51 5.14

Phenols 3.04 2.10

Acetic acid 0.58 0.13

Furfural 0.40 n.d.

Hydroxymethylfurfural 0.09 n.d.

n.d. not detectedaMean values of two determinations

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where two periods of ethanol fermentation were clearly visi-ble, and the highest specific rates of ethanol production wereachieved with glucose alone [30].

Table 2 summarizes the fermentation parameters of P.stipitis grown in sunflower meal hemicellulosic hydrolysateunder three different rotations. The highest values of thefermentative parameters were observed in the condition inwhich sugar consumption and cell growth were favored(200 rpm). The maximum values of final ethanol yield basedon glucose plus xylose (YP/S 0.23 g/g), biomass yield based onglucose plus xylose (YX/S 0.11 g/g), volumetric ethanol pro-ductivity (QP 0.12 g/L h), and efficiency percentage based ontheory (44.73 %) were found at 200 rpm. The results of thisstudy are similar to those obtained byNigam [7] (YP/S 0.25 g/gand QP 0.10 g/L h) with a parent culture of P. stipitis NRRLY-7124 grown in hardwood acid hydrolysate. When P. stipitisNRRL Y-7124 was grown in sunflower hull hydrolysate,ethanol yield YP/S was 0.32 g/g and QP 0.065 g/L h [19].

However, higher values were obtained with P. stipitisNRRL Y-7124 in a bioreactor tank using a semidefined me-dium (YP/S 0.32 g/g and QP 0.32 g/L h) [29] and even in

shaking bath fermentations with rice straw hemicellulosichydrolysate (YP/S 0.37 g/g and QP 0.39 g/L h) [34].

In the ethanol production from sugar maple hemicellulosichydrolysate by P. stipitis strain NRRL Y-11543, it was ob-served that the time it takes to reach the maximum ethanolproduction was affected by the agitation, because the maxi-mum ethanol concentration was reached after only 68.5 h at300 rpm, while the ethanol concentration continued to powerincrease even after 130 h of fermentation when the agitationrate was set at 50 rpm [35].

In the present work, the initial pH which was approximate-ly 5 may have enhanced the inhibition potential of acetic acidpresent in the medium. The toxic effect of acetic acid isprimarily a function of the undissociated acetic acid concen-tration and is pH dependent. With P. stipitis CISR-Y633(CBS 7126), at pH 6.5, only acetic acid concentrations greaterthan 4 g/L reduced the amount of ethanol produced; however,at pH 5.1, 1.0 g/L of acetic acid reduced ethanol concentrationby 50 % [36]. With P. stipitis DSM 3651, 3.5 g/L acetic acidin a medium at pH 5.0 inhibited completely both growth andethanol production [31].

Fig. 1 Variation of theconcentration of biomass,glucose, and xylose and ethanolproduction during the growth ofP. stipitis in sunflower mealhemicellulosic hydrolysate underdifferent agitations: 100 (a), 150(b), and 200 rpm (c)

Table 2 Final fermentative parameters of ethanol production by the yeast P. stipitis ATCC 58376 during cultivation in sunflower meal hemicellulosichydrolysate at 100, 150, and 200 rpm

Agitation (rpm) YP/S (glucose + xylose) (g/g) YX/S (glucose + xylose) (g/g) QP (g/L h) η (%) X f (%) Sxil (%)

100 0.12 0.034 0.07 25.02 76.88 6.50

150 0.17 0.080 0.09 32.95 89.16 4.11

200 0.23 0.124 0.12 44.73 91.40 7.64

YP/S ethanol yield, YX/S cell yield,QP ethanol volumetric productivity, η ethanol total yield efficiency, Xf percentage of cells produced, Sxil percentage ofxylose remaining

Biomass Conv. Bioref.

In a previous work with P. stipitis ATCC 58376 [30], adecrease in both total substrate consumption rate and ethanolproduction rate was attributed in part to the accumulation ofethanol in the medium. However, up to now, there are noreports on the ethanol tolerance and the maximum criticalethanol concentration for this yeast strain.

Regarding xylitol production (Fig. 2a), the effect of agita-tion was the opposite to that observed for ethanol production,since at 100 and 150 rpm, the concentrations of xylitol obtainedwere greater than at 200 rpm. The maximum production ofxylitol (1.48 g/L) occurred at 100 rpm after 72 h. Our results arein agreement with those observed with P. stipitis NRRL Y-7124 during conversion of the sugar maple hemicellulosichydrolysate into ethanol. Xylitol concentration was reducedwith increasing aeration which enabled the yeast to divertadditional carbon to ethanol formation (up to 150 rpm) [33].

In a study performed to evaluate the effect of ethanolduring fermentation of P. stipitis NRRL Y-7124, it was ob-served that the yield of xylitol increased linearly with theincrease of ethanol concentration, and as a consequence, theethanol yield declined proportionally. The authors suggestedthat ethanol may cause a disturbed NAD+/NADH2 balanceduring anaerobic xylose metabolism [37].

Besides xylitol, other by-products formed during the fer-mentation process such as acetic acid and glycerol were de-tected in the medium (Fig. 2a). The acetic acid concentrationinitially present in the medium increased during the first 24 hwith the agitations of 150 and 200 rpm followed by a reductionuntil the end of the fermentative process. With the agitation of100 rpm, pH increased more slowly followed by a slightdecrease. The highest concentration of acetic acid (0.23 g/L)

was observed after 24 h at 200 rpm. The lowest concentration(0.06 g/L) was also observed at 200 rpm at the end of fermen-tation (72 h), which indicates that the increase of aeration had astrong correlation with the acetic acid metabolism in the yeast.

Concerning pH variation in the fermentations, a pH de-crease during the first 24 h for all the three tested agitationswas observed; however, in the tests at 100 rpm, pH decreasewas extended to 48 h, followed by an increase until the end ofthe fermentation. In the fermentations conducted at 150 and200 rpm, pH increase initiated after a shorter period of 24 h.The pH increase can be attributed to the consumption of aceticacid [38, 39]. In fact, by correlating pH variation (Fig. 2d)with the variation of acetic acid concentration (Fig. 2c), it canbe seen that the increase of acetic acid concentration coincideswith the decrease of pH of the medium.

During the fermentation of sunflower hull hydrolysateby P. stipitis NRRL Y-7124 under uncontrolled pH oper-ation, pH had, firstly, a tendency to decrease followed bya slow increase at the second stage of fermentation. Withincreasing air supply, an increase in pH took place in allruns [18], similar to the behavior observed with P. stipitisATCC 58376 in this work.

With respect to glycerol, the production rate was similarduring the first 24 h for all the three conditions tested, but fromthat time, the results were influenced by the agitation, with amaximum production (1.15 g/L) attained at 200 rpm, thecondition in which the highest cell growth and ethanol pro-duction were observed. The formation of glycerol has beenobserved as a response of the stress conditions to the yeast,normally associated with the presence of toxic compoundspresent in the hemicellulosic hydrolysate [40]. Thus, it was

Fig. 2 Variation of theconcentration of xylitol(a), glycerol (b), acetic acid(c), and pH (d) during thecultivation of P. stipitis ATCC58376 in sunflower mealhemicellulosic hydrolysate at 100(dotted line and open diamonds),150 (dotted line and closeddiamonds), and 200 (solid lineand closed diamonds) rpm

Biomass Conv. Bioref.

expected in this study due to the presence of phenolics andacetic acid in the hydrolysate even after detoxification.

4 Conclusions

The high concentrations of sugars and moderate levels ofinhibitors demonstrate that the sunflower meal hemicellulosichydrolysate can be a good alternative for bioethanol produc-tion. Furthermore, the possibility of using the hydrolysatewithout a previous concentration step may contribute to theeconomic viability of the process. The establishment of moreadequate conditions is necessary to improve the fermentabilityof P. stipitis ATCC 58376 in sunflower hydrolysate. On theother hand, other strains with a better performance in hydro-lysate fermentations should be also tested.

Acknowledgments The authors acknowledge the company CaramuruSA for providing the sunflower meal and the Brazilian National ResearchCouncil (CNPq) and Itaipu Technological Park (PTI) for master’s degreescholarship to Danielle Camargo.

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