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Assessment of sweet potato [Ipomoea batatas (L.)Lam] for bioethanol production in southern ItalyA. Montefuscoa, M. Duranteb, S. Grassia, G. Piroa, G. Dalessandroa & M. S. Lenucciaa Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (Di.S.Te.B.A.), Universitàdel Salento, via prov.le Lecce-Monteroni, 73100 Lecce, Italyb CNR-ISPA Sez. Lecce, via prov.le Lecce-Monteroni, 73100 Lecce, ItalyAccepted author version posted online: 16 Sep 2014.Published online: 09 Oct 2014.

To cite this article: A. Montefusco, M. Durante, S. Grassi, G. Piro, G. Dalessandro & M. S. Lenucci (2014): Assessmentof sweet potato [Ipomoea batatas (L.) Lam] for bioethanol production in southern Italy, Plant Biosystems - AnInternational Journal Dealing with all Aspects of Plant Biology: Official Journal of the Societa Botanica Italiana, DOI:10.1080/11263504.2014.965799

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ORIGINAL ARTICLE

Assessment of sweet potato [Ipomoea batatas (L.) Lam] for bioethanolproduction in southern Italy

A. MONTEFUSCO1,*, M. DURANTE2,†, S. GRASSI1,‡, G. PIRO1,{, G. DALESSANDRO1,§, &

M. S. LENUCCI1

1Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (Di.S.Te.B.A.), Universita del Salento, via prov.le Lecce-

Monteroni, 73100 Lecce, Italy and 2CNR-ISPA Sez. Lecce, via prov.le Lecce-Monteroni, 73100 Lecce, Italy

AbstractThe potential of sweet potato as an alternative crop for bioethanol production has been assessed. We evaluated the amount ofsoluble sugars, starch and cell wall polysaccharides in tubers of three sweet potato cultivars characterized by different pulpand peel colouration: “Yellow yam” with yellow flesh and brown peel, “White yam” with white flesh and white peel and“Orange yam” with orange flesh and brown peel. The results confirm the high concentration of carbohydrates in sweet potatotubers, especially “Yellow yam”, mainly in the form of starch (67%) and soluble sugars (26%). “Yellow yam”, which is themost widespread cultivar in Salento, appeared the best choice as biomass for bioethanol production. It is characterized byhigh productivity (20–40 tons/ha year). Results also suggest that “Yellow yam” cultivar has great potential as a bioethanolsource in southern Italy with an estimated agroindustrial production yield higher than 2032 l/ha year.

Keywords: Carbohydrates, cell wall glycosyl composition, starch biomass, sugar fermentation, thermostable a-mylases

Abbreviations: AIR, alcohol insoluble residue; DE, dextrose equivalent; DNS, dinitrosalicylic acid; HPAEC-PAD,high performance anion exchange chromatography with pulsed amperometric detection

Introduction

Although the technologies for energy production

from ligno-cellulosic biomass are improving, at the

moment, the fermentation of starch biomass rep-

resents the most realistic and immediate response to

the inexorable rise in energy demand in all sectors of

human activities and, particularly, for bioethanol

production. At the current growth rate (þ30% per

year), world bioethanol production will exceed 780

£ 109 l in 2020 (Biofuels Platform Report 2011).

These data generate a growing concern due to the

use of food crops, such as maize and wheat, for

bioethanol production, causing the increase of food

prices and exacerbating world hunger.

In this context, it is important to assess the

possibility of using other starchy biomasses to

produce bioethanol at the local level. Sweet potato

[Ipomoea batatas (L.) Lam.] ranked fifth among food

crops in developing countries after rice, wheat, maize

and cassava, but its use as food is marginal in

industrialized countries. In Italy, sweet potato is

produced mainly in Emilia Romagna, Lazio and

Apulia. In Apulia, sweet potato is cultivated in the

provinces of Lecce, Bari and Brindisi. Although

sweet potato is commonly considered a “small

farmer’s crop”, if cultivated on large areas, it

produces high amount of starch biomass at low

cost. Production of tubers varies from 15 to 30 tons/

ha, but it is possible to improve these values with

more oriented cultivation techniques. The high

starch content of tubers (,17.5% fresh weight) is

one of the features that makes sweet potato

interesting for ethanol production. On a dry weight

basis, sweet potato crop produces 40–50% starch,

and this production is higher than maize, wheat and

potatoes and allows the production of more ethanol

per hectare than other crops (maize, sugar cane and

sugar beet) that are considered as energy crops (Ziska

et al. 2009; Lee et al. 2012; Duvernay et al. 2013).

In addition, the high environment adaptability of

q 2014 Societa Botanica Italiana

Correspondence: M. S. Lenucci, Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (Di.S.Te.B.A.), Universita del Salento, via prov.le Lecce-

Monteroni, 73100 Lecce, Italy. Tel: þ39 0832 298612. Fax: þ39 0832 298858. Email: marcello.lenucci@unisalento.it

Plant Biosystems, 2014http://dx.doi.org/10.1080/11263504.2014.965799

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sweet potato, which grows well in poor soils and

requires little cultural care, makes it easy to grow on

underutilized marginal soils by turning them into

energy fields.

Although the technology for starch biomass

conversion to bioethanol is widely established

(Gomez et al. 2008), it is necessary to optimize the

process based on the characteristics of the particular

biomass, taking into account any varietal character-

istics. Consequently, in the last few years, the

potential of sweet potato for bioethanol production

has been largely investigated by many authors at

laboratory, pilot and industrial scales and different

simple or simultaneous chemical, enzymatic or

microbiological saccharification and fermentation

procedures have been optimized according to

cultivar characteristics, territory resources or econ-

omic convenience (Zhang et al. 2010, 2011; Lee

et al. 2012; Duvernay et al. 2013; Lareo et al. 2013;

Swain et al. 2013). The aim of this work was to

evaluate the potential use of sweet potato as an

energy crop in southern Italy marginal soils and to

identify good practice at laboratory scale for the

production of bioethanol from this biomass. With

this paper, we hope to contribute significantly to

increase the knowledge regarding the potential use of

sweet potato tubers for bioethanol production.

Materials and methods

Chemicals

All the sugars used as standards were from Fluka

(Buchs, Switzerland). All other chemicals were

purchased from Sigma Chemical Co. (St. Louis,

MO, USA).

Sweet potato cultivation

The analyses were carried out on tubers of three

sweet potato cultivars obtained from local farmers,

phenotypically different for the flesh and peel

colouration. The cultivars were named on the basis

of their flesh colour: “Yellow yam”, with yellow flesh

and brown peel; “White yam”, with white flesh and

white peel; “Orange yam”, with orange flesh and

brown peel (Supplementary Figure 1). All cultivars

were grown simultaneously in an open-field in the

province of Lecce (Salice Salentino), southern Italy,

on a well-drained sandy soil. It lies at a latitude of

408230N and 178570E longitude. It has an altitude of

,50m above the sea level and an average annual

rainfall of 683mm.

Sprouts were obtained by planting small or

medium-sized roots close together in nursery beds.

When resulting sprouts reached 22–30 cm in length,

they were removed from the storage tubers and

transplanted into the field with an in-row spacing of

20 cm and a between-row spacing of 80 cm. Three

parcels of 8m2 were used, each containing 50 plants

per cultivar. All agronomic practices such as

weeding, earthing up and other routine activities

were conducted as per the recommendation set for

the crop. Fertilization was not applied. Harvesting

was done after 4 months when physiological maturity

and complete tuber development attained.

Sweet potato flour preparation

About 5 kg of sweet potato tubers for each cultivar

and from each parcel were randomly selected,

washed, dried, cut into small pieces without

removing the peel and homogenized in a blender

(Waring Laboratory, Torrington, CT, USA). The

homogenate was immediately frozen with liquid

nitrogen and lyophilized in a freeze dryer ALPHA

2–4 LSC Christ (Martin Christ Gefriertrocknung-

sanlagen GmbH, Osterode am Harz, Germany) to a

constant weight. The lyophilized material was

ground to 500mm by a laboratory mill (Retsch

GmbH, Haan, Germany) to obtain a homogeneous

powder (sweet potato flour).

Soluble sugar extraction

Triplicate 2 g aliquots of the flour (9 samples per

cultivar) were extracted with 50ml of 60% ethanol

(v/v) at 48C, under constant shaking (180 rpm),

overnight. The suspension was vacuum filtered on a

Buchner funnel and the alcohol insoluble residue

(AIR) was re-extracted with 50ml of 60% ethanol

(v/v) in the same conditions. The filtrates were

combined and dried in a Model R-120 Rotavapor

(Buchi Italy Srl, Assago, Milan, Italy) at 408C. The

dried material was dissolved in distilled water to a

final volume of 100ml and assayed by high

performance anion exchange chromatography with

pulsed amperometric detection (HPAEC-PAD) as

described in the “HPAEC-PAD protocol” section.

Starch determination

Three aliquots (100mg) of the AIR, previously oven-

dried at 708C to a constant weight, were used for

starch determination. Each aliquot was incubated

with 40U of a-amylase (from Aspergillus oryzae,

1000U/ml, Megazyme International Ireland Ltd,

Bray, Ireland) (EC 3.2.1.1) and 40U of amyloglu-

cosidase (Aspergillus niger, 1000U/ml, Megazyme

International Ireland Ltd) (EC 3.2.1.3) in 4ml of

50mM Na-acetate buffer, pH 5.5 containing 5mM

CaCl2, at 508C for 24 h, under continuous stirring.

At the end of the incubation period, undigested

polysaccharides were precipitated in 70% ethanol

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(final concentration) by centrifuging at 12,100g for

10min at 28C in a Biofuge 15 Heraeus Sepatech

bench centrifuge (Heraeus Sepatech GmbH, Oster-

ode, Germany). Digestion was repeated until starch

was undetectable in the pellet by staining small

aliquots with a potassium iodide–iodine solution

(usually two or three digestion treatments were

sufficient). The hydrolysates were combined

together, vacuum dried and dissolved in 5ml of

distilled water. Aliquots were opportunely diluted

and assayed by HPAEC-PAD.

Cell wall polysaccharide glycosyl analysis

The remaining de-starched pellet was extensively

digested with 5U/ml pronase in Tris–HCl 50mM,

pH 7.0, for 24h at 378C, under constant stirring.

Undigested material, mainly constituted of cell wall

polysaccharides (pectins, hemicelluloses and cellu-

lose), was recovered by centrifugation at 5000g for

10 min, washed twice with diethyl ether (5ml) and

dried in an oven at 708C to constant weight. Three

aliquots (2mg) of this material were hydrolysed with

1ml of 2N trifluoroacetic acid (TFA), in flame-sealed

bottles at 1218C, 1 atm, for 90min. Samples were then

centrifuged (17,400g; 10min) and the obtained

supernatants were vacuum-dried at 408C, re-dissolved

in1mlofdistilledwater andanalysedbyHPAEC-PAD.

The 2N TFA resistant residue (mainly cellulose)

was hydrolysed in 3% H2SO4 at 1218C for 90min as

described by Piro et al. (2000). After neutralizing the

acid with NaOH, samples were centrifuged at

17,400g for 10min. The supernatant was recovered,

filtered and analysed by HPAEC-PAD.

HPAEC-PAD protocol

All samples were filtered through a 0.2mm Spartan

13 filter (Schleicher & Schuell Microscience, Dassel,

Germany) and then analysed on a Dionex HPAEC

with a CarboPac PA10 column as described

by Lenucci et al. (2008). The eluent flow rate was

1ml/min at room temperature, and 20ml samples

were injected. Sugars were quantified with a pulsed

amperometric detector with gold electrode. Mono-

saccharide separation was performed by using the

following eluents (gradients were linear at all

specified time points): 0–20min, 0.02M NaOH;

20.1–27min, 0.20M NaOH; 27.1–31min, 0.80M

NaOH; 31.1–42min, 0.02M NaOH.

The inter- and intra-day variability of the method

was measured repeating the analyses three times on

the same day and three times on three consecutive

days using a mixture of the authentic markers

(arabinose, fructose, galactose, galacturonic ac.,

glucose, glucuronic ac., maltose, mannose, rham-

nose, sucrose and xylose). The coefficients of

variation of the intra- and inter-day variability were

calculated to be below 5% and 7%, respectively.

Optimization of sweet potato starch gelatinization and

liquefaction processes

Tests were conducted using two different gelatiniza-

tion and liquefaction thermostable a-amylases:

Termamyl 120 L (from Bacillus licheniformis,

20,000U/ml, Novozyme, Bagsvaerd, Denmark)

and Ban 240L (from Bacillus amyloliquefaciens,

$250U/g, Novozyme). Increasing concentrations

of lyophilized sweet potato biomass (15%, 20%,

25%, 30%, 35%w/v) were prepared by suspending

the appropriate amount of sweet potato flour in 1ml

of 0.2M Na-phosphate buffer containing 70 ppm

CaCl2 at pH 6.0. When Ban 240L was used, starch

was previously gelatinized by autoclaving at 1218Cfor 20min and then liquefied with 5 or 10U/ml of the

enzyme, at 708C in a water bath. Alternatively,

Termamyl (5 or 10U/ml) was directly added to

starchy substrates and the simultaneous gelification/

liquefaction process was conducted at 1058C in

autoclave. The amount of reducing sugars released in

solution was monitored over time (from 15 to

240min) by the dinitrosalicylic acid (DNS) method

(Bailey et al. 1992).

Optimization of sweet potato starch saccharification

process

The samples liquefied with Termamyl were sequen-

tially saccharified with different concentrations (from

0 to 30U/ml) of amyloglucosidase (from Aspergillus

niger, 1000U/ml, Megazyme International Ireland

Ltd) at 508C, for 24 h, under constant stirring

(180 rpm). The undigested material was precipitated

by centrifugation at 12,100g for 10min. The

reducing sugar content was determined using the

DNS method (Bailey et al. 1992). Tests were also

carried out reducing the incubation times.

Fermentation tests

The sugar syrup obtained after sweet potato starch

saccharification was opportunely diluted (by adding

distilled water) or vacuum concentrated and sub-

jected to fermentation with a selected Saccharomyces

cerevisiae strain (CISPA 161) immobilized in sodium

alginate beads. Sodium alginate beads were prepared

as previously described (Lenucci et al. 2013). Briefly,

cells were suspended in 100ml of 3% (w/v) sodium

alginate aqueous solution. The slurry was poured

drop-by-drop, from a hypodermic needle using a

peristaltic pump (Miniplus 3, Gilson, Inc., Mid-

dleton, WI, USA), into 100ml of 0.05M CaCl2 with

constant stirring, at room temperature and under

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sterile conditions. The produced spherical beads

(approx. 3.8mm diameter) were washed in distilled

water and immediately used. For each substrate

(100ml), 700 beads, approx. 70 £ 1012 yeast cells,

were used. The consumption of sugars and pro-

duction of bioethanol were monitored over time by

an HPLC system (Agilent 1100 series) equipped

with a refractometric detector and an Aminex HPX-

87 H (7.8 £ 300mm) column. Separation was

performed at 558C with an eluent flow (H2O

acidified with H2SO4, pH 2.3) of 0.6mL/min. All

samples were filtered through a 0.2mm Spartan 13

filter (Schleicher & Schuell Microscience) before

being tested.

Statistical analysis

Results are presented as mean value ^ standard

deviation. Statistical analysis was based on a one-way

ANOVA test. The post hoc method by Holm-Sidak

was applied to establish significant differences

between means with a confidence level of 95%.

All statistical comparisons were performed using the

SigmaStat Version 3.11 software (Systat Software,

Inc., Chicago, IL, USA).

Results and discussion

Carbohydrate analyses were carried out on tubers of

three sweet potato cultivars characterized by differ-

ent pulp and peel colouration: “Yellow yam” with

yellow flesh and brown peel, “White yam” with white

flesh and white peel and “Orange yam” with orange

flesh and brown peel (Supplementary Figure 1).

The percentage of dry weight (%dw) of tubers

significantly varied among cultivars from

28.4 ^ 1.2% in the “Yellow yam”, 16.4 ^ 0.8% in

“White yam” and 7.6 ^ 0.6% in “Orange yam” (data

not shown) confirming the high variability reported

by Woolfe (1992) for sweet potato cultivars grown in

Taiwan (13.6–35.1%dw) and by Cereda et al.

(1982) comparing data from 18 cultivars grown in

the same area of Brazil (22.9–48.2%dw).

Soluble sugar composition of sweet potato tubers

is reported in Table I. On a fw basis, the “Yellow

yam” cultivar showed the highest amount of total

soluble sugars (,73.6 g/kg fw), followed by “Orange

yam” (,22.5 g/kg fw) and “White yam” (,15.3 g/kg

fw). On a dw basis, “Orange yam” and “Yellow yam”

cultivars showed a similar amount of total soluble

sugars (26.2% and 25.8%, respectively) which was

much lower in “White yam” cultivar (9.3%). These

values fell in the ranges reported by Bradbury and

Holloway (1988) for tubers grown in the South

Pacific (3.8–56.4/100 g dw), and by Ravindran et al.

(1995) for tubers of 16 cultivars of sweet potato

grown in Sri Lanka (3.7–9.9/100 g dw).

The qualitative composition of soluble sugars

was almost identical in the three cultivars; sucrose

was the most abundant soluble sugar, followed by

glucose and fructose. A similar pattern was reported

by other authors who also showed large variations

of sucrose content among cultivars (Kawabata

et al. 1984; Picha 1985; Troung et al. 1986). In the

three cultivars, genotype-related differences in sugar

molar ratios were evidenced. Small quantities of

maltose were detected in “Yellow yam” (2.2 g/kg fw;

1.0mol%) and “White yam” cultivars (0.3 g/kg fw;

1.1mol%) probably deriving from starch hydrolysis

by endogenous amylases (Walter et al. 1975).

Table II shows the amount of storage and cell wall

polysaccharides of sweet potato tubers expressed as

g/kg fw and %dw. As expected, starch was the most

abundant polysaccharide in all cultivars. “White

yam” and “Yellow yam” showed starch content

(79.2%dw and 64.6%dw, respectively) in line with

the average of the specie (72%dw) (Ravindran et al.

1995), while “Orange yam” cultivar showed much

lower content (24.6%dw). Differences in the amount

of starch of sweet potato tubers were previously

reported for cultivars grown in Brazil (43–79%dw)

and for Filipino-American cultivars (33–73%dw)

(Cereda et al. 1982; Troung et al. 1986). This

genetic variability may be further amplified by many

factors such as soil composition, climate, environ-

mental conditions, cultivation and agronomic tech-

niques, presence of parasites and so on (Bradbury &

Table I. Qualitative and quantitative analysis of soluble sugars in fresh tubers of three different sweet potato cultivars.

Yellow yam White yam Orange yam

Soluble sugars g/kg fw %dw mol% g/kg fw %dw mol% g/kg fw %dw mol%

Glucose 15.7^ 1.2 5.5 22.0^ 1.2 2.0^ 0.1 1.2 11.0^ 0.1 6.6 ^ 0.5 8.7 28.0 ^ 0.1

Fructose 12.3^ 1.0 4.3 17.0^ 1.0 2.0^ 0.1 1.2 8.0^ 0.1 6.7 ^ 1.7 8.8 19.0 ^ 0.7

Sucrose 43.4^ 2.7 15.2 60.0^ 2.7 11.0^ 0.2 6.7 80.0^ 0.2 9.2 ^ 1.1 12.1 53.0 ^ 1.2

Maltose 2.2^ 0.3 0.8 1.0^ 0.3 0.3^ 0.1 0.2 1.1^ 0.1 – – –

Total 73.6^ 5.2 25.8 15.3^ 0.5 9.3 22.5 ^ 3.3 29.6

Notes: Values are expressed as g/kg fw, %dw and mol% with respect to total identified sugars. The data represent the mean ^ standarddeviation of nine independent replicates (n ¼ 9).

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Holloway 1988). A very small amount of inulin

(,0.5 g/kg fw; 0.2%dw) was detected only in the

“Yellow yam” cultivar.

Cell wall polysaccharides contributed to tuber dw

but to a different extent among cultivars (,4.0%dw

in “Yellow yam”; ,6.5%dw in “White yam” and

,8.5%dw in “Orange yam”). Even the ratio between

cellulose and matrix polysaccharides (pectins and

hemicelluloses) was cultivar-dependent. In the “Yel-

low yam” cultivar, the amount of cellulose (6.1 g/kg

fw; 2.1%dw) was slightly higher than matrix

polysaccharides (5.3 g/kg fw; 1.9%dw) with a ratio

of ,0.90. Cellulose was less abundant than matrix

polysaccharides in “Orange yam” (cellulose/matrix

polysaccharide ¼ 0.41) and “White yam” (cellulose/

matrix polysaccharide ¼ 0.27) cultivars.

The monosaccharide composition of matrix

polysaccharides, obtained by 2N TFA hydrolysis of

purified walls of sweet potato tuber parenchyma

cells, was similar in the three cultivars; however,

significant differences in the mol% and ranking of the

released monosaccharide were detected (Table III).

In all the cultivars, the mole percent of galacturonic

acid exceeded that of rhamnose, thus indicating the

presence of pectins constituted of rhamnogalactur-

onan I (RG I) and homogalacturonan domains. The

“Orange yam” cultivar had a higher rhamnose/

galacturonic acid ratio (0.66) than those of “White

yam” (0.59) and “Yellow yam” (0.53). Since the

backbone of RG I consist of alternating rhamnose

and galacturonic acid residues in a molar ratio of 1:1,

the differences in the relative proportion of those

sugars found among cultivars reflect the contribution

of RG I to pectins. Hence, with a rough approxi-

mation due to the considerable uncertainty about the

recovery of rhamnose and galacturonic acid residues

after TFA hydrolysis, “Orange yam” pectins were

found to contain a higher percentage of RG I

domains (80%) than those of “White yam” (75%)

and “Yellow yam” (69%). Galactose and arbinose

were indicative of the presence of neutral RG I-side

chains of arabans, galactans and/or arabinogalattans,

confirming the reports of Noda et al. (1994) who

found that sweet potato pectic substance mainly

composed of uronic acid (47.1%) and galactose as

the predominant neutral sugar residues. Due to the

differences in arabinose and galactose contents, the

length and/or degree of substitution of RG I

backbone seems to vary among cultivars. The

presence of high mol% of xylose (24.9mol% in

“Yellow yam”, 20.0 mol% in “White yam” and

13.1mol% in “Orange yam”) and glucose (18.5mol

% in “Yellow yam”, 18.0mol% in “White yam” and

12.0mol% in “Orange yam”) indicates the presence

of xyloglucans and xylans, the main hemicellulosic

component of sweet potato tubers, in accordance

with the findings of Noda et al. (1994) and Salvador

et al. (2000). Mannose was detected in a molar ratio

ranging between 5.1 and 10.6mol%, suggesting the

presence of small amounts of glucomannans and/or

high-mannosidic glycoproteins.

Due to its good characteristics in terms of

percentage of dry weight (28.4%dw), soluble sugar

(25.8%dw) and starch (64.6%dw) contents, “Yellow

yam” cultivar appeared the best choice as biomass

for bioethanol production. This cultivar, which is

the most popular in the Salento area, is also

Table III. Glycosyl residue composition of matrix polysacchar-

ides (pectins and hemicelluloses) obtained by 2N TFA hydrolysis

of purified cell walls from three different sweet potato cultivars.

Sugars

Yellow yam

(mol%)

White yam

(mol%)

Orange yam

(mol%)

Rhamnose 5.0^ 0.6 3.8^ 0.7 8.6^ 0.2

Arabinose 9.5^ 0.5 19.0^ 1.2 15.0^ 0.4

Galactose 22.0^ 2.9 25.0^ 0.3 31.0^ 0.2

Glucose 18.5^ 0.6 18.0^ 3.5 12.0^ 1.2

Mannose 10.6^ 0.9 7.6^ 1.6 5.1^ 0.4

Xylose 24.9^ 2.0 20.4^ 4.3 13.1^ 0.9

Galacturonic Ac. 9.5^ 0.6 6.4^ 0.9 13.0^ 0.8

Total (mg/g fw) 5.3^ 0.5 8.3^ 0.1 4.6^ 0.7

Notes: Values are expressed as mol%with respect to the total of theidentified sugars. The data represent the mean ^ standarddeviation of nine independent replicates (n ¼ 9).

Table II. Content of storage (starch and inulin) and cell wall polysaccharides (matrix polysaccharides and cellulose) in three sweet potato

cultivars.

Yellow yam White yam Orange yam

g/kg fw %dw g/kg fw %dw g/kg fw %dw

Storage polysaccharides

Starch 183.5^ 6.5 64.6 130.2 ^ 15.2 79.2 18.7 ^ 2.4 24.6

Inulin 0.5^ 0.2 0.2 – – – –

Cell wall polysaccharides

Matrix polysaccharides 5.3^ 1.5 1.9 8.3 ^ 0.1 5.0 4.6 ^ 0.7 6.0

Cellulose 6.1^ 0.6 2.1 2.3 ^ 0.2 1.4 1.9 ^ 0.1 2.5

Total polysaccharides 195.4^ 8.8 68.6 164.8 ^ 15.5 85.7 25.2 ^ 3.2 33.1

Notes: Values are expressed as g/kg dw and %dw. The data represent the mean ^ standard deviation of nine independent replicates (n ¼ 9).

Bioethanol from sweet potato tubers 5

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characterized by high productivity (20–40 tons/ha).

These features make “Yellow yam” cultivar competi-

tive with industrial sweet potato breeding lines

specifically developed to produce bioethanol

(Duvernay et al. 2013; Lareo et al. 2013). Using

this cultivar, we performed tests to optimize the

processes of starch hydrolysis to glucose. The

conversion of starch biomass into glucose is in fact

a crucial point since fermenting microorganisms,

such as Saccharomyces cerevisiae and Zymomonas

mobilis, do not possess amylolytic activities (Ang et al.

2001). Starch enzymatic hydrolysis is generally

preferred over chemical hydrolysis for the high

substrate-selectivity, since ease of use along with the

possibility for reuse of the enzymes help to reduce

costs and ensure high safety standards. Starch

enzyme hydrolysis consists of three steps: gelatiniza-

tion, liquefaction and saccharification. At 50–608C,

starch granules swell and absorb water forming a gel;

during liquefaction, the addition of exogenous a- andb-amylases catalyse starch hydrolysis into glucose,

maltose and a- and b-limit dextrins. Finally, during

saccharification, a-1,6- and a-1,4-amyloglucosidase

hydrolyse maltose, and dextrins turn into glucose

which represents the substrate that can be easily

fermented to ethanol.

In this work, two hydrolytic commercial thermo-

stable a-amylases were used: Termamyl and Ban.

Termamyl allows the simultaneous gelatinization and

liquefaction of starch at high temperatures (1058C),

whereasBan requires an initial gelatinization at 1218Cfollowed by liquefaction at 708C. The hydrolytic

activity of each enzyme, in relation to percentage of

starch biomass, time and enzyme concentration, was

compared by dextrose equivalent (DE), a parameter

measuring the amount of reducing sugars released

during hydrolysis. By convention, DE of native starch

is zero while glucose has a DE of 100. Usually,

liquefaction leads to the formation of products with

DE between 10 and 40, whereas saccharification

products have DE between 40 and 98 depending on

the operative conditions (Kennedy et al. 1988).

Termamyl (10U/ml) efficiently hydrolysed substrates

with a starch biomass concentration of 30% w/v in

30–45min (DE . 40) (Figure 1). By using Ban

(10U/ml), values higher than 40 DE were obtained

only when starch biomass was less than 25% w/v and

at a time range from 45 to 60min. Halving Termamyl

concentration to 5U/ml, DE higher than 40 were

obtained at starch biomass concentrations up to 25%

w/v, after at least 30min. The same hydrolysis

efficiency was obtained at starch biomass concen-

tration below 20% w/v, after 45–60min, with Ban.

Differently fromTermamyl, using Ban, gelatinization

and liquefaction did not occur simultaneously, so that

the time of starch hydrolysis was always much longer.

15

45120

0

20

40

60

80

100

15 20 25 30 35

D.E

.

Starch biomass (%)

5 U/mL

15

45120

0

20

40

60

80

100

15 20 25 30 35

D.E

.

Starch biomass (%)

10 U/mL

Termamyl

15

45120

0

20

40

60

80

100

15 20 25 30 35

D.E

.

Starch biomass (%)

5 U/mL

15

45120

0

20

40

60

80

100

15 20 25 30 35

D.E

.

Starch biomass (%)

10 U/mL

Ban

20–40D.E. 40–60 D.E. 60–80 D.E.

Figure 1. Effect of Termamyl and Ban concentrations, substrate concentrations (15%, 20%, 25%, 30%, 35%w/v) and incubation time on the

amount of DE obtained during sweet potato starch gelatinization/liquefaction. The data represent the mean of three independent replicates

(n ¼ 3).

6 A. Montefusco et al.

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Our data clearly show that Termamyl was more

efficient than Ban to catalyse Ipomoea batatas starch

liquefaction. Much high values of starch hydrolysis

(78–80% and 61–74% for fresh sweet potato and

flour, respectively) were reported by Lareo et al.

(2013) possibly due to improved starch digestibility of

the cultivar K 9807.1 specifically developed for

bioenergy purposes and/or to n higher efficiency of

Liquozyme compared to Termamyl and Ban.

In order to strictly control biomass concen-

tration, gelatinizaton/liquefaction tests were per-

formed on the freeze-dried sweet potato

homogenate (sweet potato flour) reconstituted by

adding the appropriate amount of water. Never-

theless, the use of fresh sweet potato homogenates of

White yam, whose percentage of dry weight was

approximately 30%, gave even better DE values in

the same operative conditions (data not shown).

A reduction of the gelatinization/liquefaction tem-

perature from 105 to 808C did not affect the DE

values of the hydrolytic products when 5U/ml

Termamyl were used (Figure 2(A)).

Samples of sweet potato gelatinized/liquefied at

808C for 45min with Termamyl were subjected to

saccharification with decreasing concentrations of

amyloglucosidase (from Aspergillus niger) at 508C for

24 h (Figure 2(B)). No significant differences

(P , 0.05) in DE values were obtained with

concentrations between 30 and 0.5U/ml amyloglu-

cosidase. DE remained almost unchanged (approxi-

mately 95–98) when saccharification time was

reduced from 24 to 2 h (data not shown).

Using the optimized conditions for sweet potato

starch hydrolysis (gelatinization/liquefaction: 30% w/v

starch biomass, 5U/ml Termamyl, 808C, 45min;

saccharification: 1U/ml amyloglucosidase, 508C,

2 h), a sugar-rich syrup was prepared. Different

concentrations of the syrup, containing from 62.0 to

267.4 g/l of fermentable sugars, were subjected to

fermentation using Saccharomyces cerevisiae (strain

CISPA 161) immobilized in sodium alginate beads

(ø ¼ 3.8mm) without the addition of any other

growth factor (Figure 3). When diluted substrates

were used (Figure 3(A),(B)), sugars were completely

metabolized after ,32 h of fermentation and the

amount of ethanol produced was 23.6 and 38.9 g/l,

respectively, corresponding to,75% of the theoreti-

cal yield. Increasing sugar concentation (Figure 3

(C)–(E)), led to an increase in both fermentation

yield and time.Ethanol producedat the concentration

of 53.1 g/l (Figure 3(C)) was about 84% of the

theoretical production, while the concentration of

0

20

40

60

80

100

120

30 20 5 2 1 0.5 0

D.E

.

Amyloglucosidase (U/ml)

0

10

20

30

40

50

60

70

105 80 70 60

D.E

.

Temperature (°C)

A

B

Figure 2. (A) Effect of temperature on sweet potato starch gelatinization/liquefaction process in the presence of Termamyl (5U/ml, 45min.).

(B) Effect of amyloglucosidase concentration on saccharification (508C, 24h) of Termamyl (5U/ml, 45min, 808C) liquefied starch. The data

represent the mean ^ standard deviation of three independent replicates (n ¼ 3).

Bioethanol from sweet potato tubers 7

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169.7 g/l (Figure 3(D)) gave a yield higher than 97%.

About 95% of the initial amount of fermentable

sugars was metabolized during the first 72 h when the

concentration of 189.5 g/l (Figure 3(E)) was used; the

remaining 5% required between 72 and 144 h. The

production of ethanol was 100 g/l, corresponding to

12% ethanol (v/v) and to 100% yield fermentation.

Similar results were reported by Lareo et al. (2013)

which proposed that ethanol concentration higher

than 100 g/l are toxic for the baker yeast S. cerevisiae.

Using the concentrations of 169.7 and 189.5 g/l

(Figure 3(D),(E)), ethanol yield was considerably

greater than that obtained with the other concen-

trations, possibly due to the presence in the syrup of

other substances, which, acting as nutrients for the

yeast, promote the fermentation process. It is, in fact,

known that the proper supplementation of nutrients

(nitrogen vitamins and metal ions) to the media may

improve ethanol tolerance and contribute to increase

the final ethanol concentration (Zhao & Bai 2009;

Breisha 2010; Shen et al. 2012). Besides, the use of

ethanol tolerant S. cerevisiae strains was reported to

further increase ethanol concentration up to,128 g/l

in laboratory scale trials (Zhang et al. 2011).

However, higher concentration of substrate

(sugars . 250 g/l) resulted in a marked inhibition of

the fermentation process (Figure 3(F)). These data

show that substrate concentration influences the time

required for complete sugar fermentation and the

ethanol yield.

Conclusions

Sweet potato production is a minor farmer crop in

the South of Italy. In Apulia, the annual yield was

approximately 600 tons (year 2010) in an area of

about 40 ha of marginal land, mainly concentrated in

the province of Lecce (ISTAT Data 2010). The

productivity of the most widely diffused cultivar,

“Yellow yam”, is approx. 20 tons/ha. This very good

yield together with a high content of starch and

soluble sugars, in line with the average of the specie,

made this cultivar an interesting source of fermen-

table sugars for bioethanol production.

0

10

20

30

40

50

60

70

0 10 20 30 40 50

Con

cent

rati

on (

g/L

)

Time (h)

0

20

40

60

80

100

120

0 20 40 60 80

Con

cent

rati

on (

g/L

)

Time (h)

0

20

40

60

80

100

120

140

0 20 40 60 80

Con

cent

rati

on (

g/L

)

Time (h)

020406080

100120140160180

0 20 40 60 80 100 120 140 160

Con

cent

rati

on (

g/L

)Time (h)

0

50

100

150

200

0 20 40 60 80 100 120 140 160

Con

cent

rati

on (

g/L

)

Time (h)

0

50

100

150

200

250

300

0 20 40 60 80 100 120 140 160 180

Con

cent

rati

on (

g/L

)

Time (h)

31.7 g/L 50.0 g/L

63.2 g/L 86.8 g/L

96.9 g/L 136.4 g/L

A B

C D

E F

Figure 3. Time course of glucose consumption and ethanol production in substrates with different concentrations of sugars, prepared

by enzymatic hydrolysis of sweet potato tubers. Dotted line: glucose; solid line: ethanol; line points and lines: theoretical yield of ethanol.

The data represent the average of three independent replicates (n ¼ 3).

8 A. Montefusco et al.

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The use of a thermo-stable amylase (Termamyl)

made the process of starch gelatinization and

liquefaction extremely efficient; moreover, a treat-

ment with reduced amounts of amyloglucosidase

allowed liquefied starch conversion into a sugar syrup

with DE close to 100. The appropriately concen-

trated syrup was efficiently fermented to ethanol

from Saccharomyces cerevisiae with a maximum

estimated industrial yield of ethanol equivalent to

,381 l/ton sweet potato dry matter per annum and

an agroindustrial yield of ,2032 l/ha per annum

(calculated based on ethanol produced in the optimal

operative conditions, local crop yield and a distilla-

tion and dehydration efficiency of 0.98).

It is important to underline that the ethanol

production can be further increased by fermenting

the ligno-cellulosic biomass of the entire plant (stalks

and leaves) that contribute to the whole productivity

of the plant. It is also interesting to consider that the

efficiency of this process can be further improved

through the use of naturally adapted or genetically

modified Saccharomyces cerevisiae strains ideally

suited for use as delivery vehicles for future bioenergy

technologies, exhibiting desirable phenotypes such as

high ethanol and cell mass production and high

temperature and oxidative stress tolerance (Argueso

et al. 2009).

The data reported in this paper show that in

southern Italy, and particularly in Apulia, Ipomoea

batatas is a high-starch biomass that can be grown in

marginal soils and used not only as a source of

carbohydrates for human consumption, but also for

bioethanol production without competing with

intensive crops for food production. At the local

level, a widespread use of biomass may ensure the

reduction of agricultural surpluses by the replacement

of traditional crops with energy crops, and would also

allow the development of new industrial initiatives

and therefore the development of marginal areas.

Acknowledgements

We thank Dr Gaetano Carrozzo for technical

assistance in sweet potato plant cultivation and Dr

Donald Ruari for improving the English style of the

manuscript.

Conflict of interest: The authors disclose any actual or

potential competing interests.

Notes

* Email: anna.montefusco@unisalento.it† Email: miriana.durante@ispa.cnr.it‡ Email: stefaniagrassi1983@libero.it{ Email: gabriella.piro@unisalento.it§ Email: giuseppe.dalessandro@unisalento.it

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