Appl Biochem Biotechnol (2014) 174:2762–2776

DOI 10.1007/s12010-014-1224-4

A Yeast Isolated from Cashew Apple Juice and Its Ability

to Produce First- and Second-Generation Ethanol

E. M. Barros & T. H. S. Rodrigues & A. D. T. Pinheiro &

A. L. Angelim & V. M. M. Melo & M. V. P. Rocha &
Luciana R. B. Gonçalves

Received: 18 July 2014 / Accepted: 5 September 2014 /

Published online: 20 September 2014
# Springer Science+Business Media New York 2014

Abstract The aim of this study was to isolate and identify an indigenous yeast from cashew
apple juice (CAJ) and then use it in the production of first- and second-generation ethanol,
using CAJ and the enzymatic hydrolysate of cashew apple bagasse (MCAB-OH), respectively.
The isolated yeast was identified as belonging to the genus Hanseniaspora. Afterward, the
effect of the medium initial pH on the production of ethanol from CAJ was evaluated in the
range of 3.0 to 5.5, with its maximum ethanol production of 42 g L−1 and YP/S of 0.44 g g−1
and 96 % efficiency. The effect of temperature (28–38 °C) on ethanol production was
evaluated in a synthetic medium, and no difference in ethanol production in the temperature
range evaluated (28–36 °C) was observed. At 32 °C, the yield, concentration, efficiency, and
productivity of ethanol when using the CAJ medium were higher when compared to the results
achieved for the synthetic medium. Regarding second-generation ethanol, the results showed
that the yeast produced 24.37 g L−1 of ethanol with an efficiency of 80.23 % and a productivity
of 4.87 g L−1 h−1 at 5 h. Therefore, Hanseniaspora sp., isolated from CAJ, is a promising
microorganism for the production of first- and second-generation ethanol.

Keywords Hanseniaspora . Ethanol . Process integration . Cashew apple juice . Cashew apple
bagasse

Abbreviations
CAJ Cashew apple juice
CAB Cashew apple bagasse
MCAB-OH Enzymatic hydrolysate of cashew apple bagasse
Pmax Maximum ethanol concentration (g L−1)
NS non-Saccharomyces yeasts

E. M. Barros : T. H. S. Rodrigues : A. D. T. Pinheiro : M. V. P. Rocha : L. R. B. Gonçalves (*)

Programa de Pós-Graduação em Engenharia Química, Universidade Federal do Ceará, Campus do Pici,
Bloco 709, 604440-554 Fortaleza, CE, Brazil
e-mail: lrg@ufc.br

A. L. Angelim : V. M. M. Melo
Departamento de Biologia, Universidade Federal do Ceará, Campus do Pici, Bloco 909,
60455-760 Fortaleza, CE, Brazil
Appl Biochem Biotechnol (2014) 174:2762–2776 2763

η Efficiency of sugar conversion to ethanol (%)

QP Ethanol volumetric productivity (g L h−1)
θS Substrate conversion (%)
YP/X Yield of product based on cell growth (g g−1)
YX/S Yield of cell growth based on substrate consumption (g g−1)
YP/S Yield of product based on substrate consumption (g g−1)
μmax Maximum specific growth rate of cells (h−1)
FPU Filter paper unit

Introduction

Nowadays, Brazil is one of the largest producers of ethanol, and it is also the country with the
highest potential for expanding its production, due to its privileged weather and availability of
soil for the large-scale production of the feedstock used in the production of this biofuel [1].
For these reasons, large companies of energy and food have invested in the sector, as well as in
the research and development of new technologies for the production of ethanol [2]. Second-
generation ethanol, produced from lignocellulosic materials, has been seen as the biofuel with
a great potential to replace fossil fuels, presenting lower environmental impacts than the first-
generation ethanol [1, 3]. Besides being abundant and having a low cost, the lignocellulosic
materials do not compete for land use with food crops [4].
One of the reasons that should make the production of ethanol increase and the costs to
decrease is the use of alternative substrates of low cost. In fact, substrate preparation is responsible
for 60–70 % of the cost to produce ethanol [5]. With this in mind, cashew apple appears as a
promising substrate, since juice and bagasse may be used to ethanol production [6, 7].
Several studies have highlighted the feasibility of using cashew apples for ethanol produc-
tion with promising results [6–10]. It is important to mention that the production of ethanol from
cashew apple cannot be considered as a substitute to the actual feedstock (sugarcane) but as a
new activity to enlarge and diversify the agribusiness. In the state of Ceará, Northeast Brazil, the
cashew (Anacardium occidentale L.) agribusiness has an important part in the local economy
based primarily on the cashew nut market [11]. On the other hand, approximately only 12 % of
the cashew apples are industrially exploited for juice production, so the largest part of the apple
production is wasted in the field [12]. Therefore, the use of the cashew apple for the production
of ethanol will not only bring economic benefits, since it is a low-cost feedstock, but will also
solve a discard problem, avoiding cashew apples to rot in the field and adding value to
the cashew market [6, 10]. A small distillery, for example, can use cashew apple as
feedstock, both cashew apple juice (CAJ) and cashew apple bagasse (CAB—a ligno-
cellulosic material), for ethanol production. Therefore, the production of second-
generation ethanol can share part of the infrastructure where the production of the
first-generation occurs (e.g., concentration, fermentation, distillation, storage, and
cogeneration facilities).
The microorganisms most commonly used in the production of ethanol come from the
Saccharomyces genus, due to their high ethanol tolerance [13, 14]. However, other microor-
ganisms are present on the surfaces of some fruits, such as grapes, mainly non-Saccharomyces
yeasts (NS), which predominate during the early stages of fermentation [14, 15]. Furthermore,
some authors observed the potential of those NS for ethanol production [16], showing
competitive values of yield and titer. One of the prevailing genera of NS yeasts that show this
potential is Hanseniaspora [16, 17]. Since the idea is to produce ethanol in small facilities in
the countryside of Brazil, the use of a strain isolated from the cashew apple itself may be an
interesting strategy to achieve a low-cost and simple process. Therefore, it is interesting to
2764 Appl Biochem Biotechnol (2014) 174:2762–2776

study the production of ethanol using this other genera of yeast in order to evaluate its
efficiency.
Apart from the medium and microorganisms, operating conditions, such as temperature and
pH, are factors that influence the effectiveness of the alcoholic fermentation. Evaluating the
effect of temperature on the kinetic parameters of fermentation is important for developing
more efficient control strategies in ethanol production industries. Although isothermal pro-
cesses are the most common in the literature [18], in industrial processes, there is no constancy
in temperature. pH is another factor that has an impact on the enzymatic activity of cells and
can modify the chemical pathways of biological reactions, as well as the kinetics of alcoholic
fermentation [19]. It is industrially important that fermentation occurs in pH range in which the
kinetics of ethanol production is favored and, also, that the ambient resists to change in pH to
maintain the stability of the process.
In this context, the aim of this work was to isolate and identify a new yeast strain from
cashew apple peduncle and then study its potential for the production of first-generation and
second-generation ethanol. The effects of temperature and pH on the kinetics of first-
generation ethanol production were investigated using CAJ as carbon source. Last but not
least, the efficiency of the selected yeast strain in the production of second-generation ethanol
was evaluated using CAB as the lignocellulosic material.

Materials and Methods

CAJ

The CAJ was kindly donated by the Brazilian Agricultural Research Corporation, Embrapa,
CE. Initially, CAJ was centrifuged at 10,000g for 15 min, and the initial concentration of
reducing sugars (glucose and fructose) was adjusted to 90 g L−1 with the addition of distilled
water. The pH of the medium was adjusted to the desired value, and subsequently, it was
sterilized by autoclaving at 110 °C for 10 min.

CAB Pretreatment

CAB was donated by Jandaia Sucos do Brazil (Ceará, Brazil), and it was pretreated according
to Rocha et al. [7]. First, a pretreatment of CAB with diluted sulfuric acid was conducted, and
then the residue was treated with 1.0 M NaOH, using a solid concentration of 7.5 % (w/v), at
121 °C for 30 min. The resulting solid was designated as hydrolysate of cashew apple bagasse
(CAB-OH).

Enzymatic Hydrolysis of CAB

CAB-OH (with particles ranging from 0.25 to 0.84 mm) was used for enzymatic hydrolysis.
The enzymatic hydrolysis was performed using a commercial enzyme Celluclast 1.5L
(Novozyme, Bagsvaerd, Denmark), with 16 % m/v CAB-OH (11.5 % w/v cellulose) and
60 FPU gcellulose−1 enzyme activity at 45 °C, 150 rpm for 72 h. After the enzymatic hydrolysis
of CAB-OH, the solid was separated by centrifugation (10,000g for 15 min), followed by
filtration. The supernatant was diluted with distilled water to the concentration of 50 g L−1
glucose and supplemented with 5 g L−1 yeast extract and 1 g L−1 (NH4)2SO4. After this, it was
sterilized at 110 °C for 10 min to be used as fermentation medium for ethanol production,
coded enzymatic hydrolysate of CAB (MCAB-OH). MCAB-OH was characterized and
Appl Biochem Biotechnol (2014) 174:2762–2776 2765

contained 85.79 g L−1 ±3.11 g L−1 of glucose and 30.34 g L−1 ±0.79 g L−1 of cellobiose, as
well as traces of xylose (2.28 g L−1 ±0.07 g L−1). No inhibitors (formic acid, acetic acid,
furfural, and hydroxymethylfurfural) were detected.

Isolation of an Ethanol-Producing Yeast from CAJ

One hundred milliliters of CAJ was added to a 250-mL sterile Erlenmeyer flask and allowed to
ferment naturally. Thereafter, 30 mL of the fermented juice was added to 120-mL yeast extract
peptone dextrose (YEPD) medium in 250-mL Erlenmeyer flask and incubated in an orbital
shaker (Tecnal TE-420, Piracicaba, Brazil) at 30 °C under agitation of 150 rpm for 24 h. After
this period, the fermented medium was centrifuged at 10 °C and 6,000g, and the precipitate was
inoculated in Sabouraud agar plates and incubated at 30 °C for 48 h. Then, the preponderant
colonies were picked and re-isolated in the Sabouraud agar to obtain pure cultures.
Afterward, YEPD complex medium, containing 10 g L−1 yeast extract, 20 g L−1 peptone, and
20 g L−1 glucose, was used for the propagation of the cells. The pH of the media was adjusted to
4.5 using 6 N HCl, and both were sterilized in an autoclave (Phoenix, Araraquara, SP, Brazil) at
110 °C for 10 min. For the maintenance of cells, YEPD medium was solidified with 20 g L−1 agar.

Molecular Identification of the Ethanol-Producing Yeast

The selected yeast strain was grown on YEPD at 25 °C and 150 rpm for 24 h. Then, 4 mL of
medium was centrifuged at 10,000g for 5 min, and the pellet of cells was washed three times
with distillated water under centrifugation. The genomic DNA was obtained by extraction of
the pellet by cetyltrimethylammonium bromide (CTAB) (Fisher Scientific, Fair Lawn, New
Jersey, USA), in accordance with the protocol described by Warner [20]. The ribosomal RNA
(rRNA) operon encompassing the 5.8S rRNA gene and the flanking internal transcribed
spacers ITS1 and ITS2 was amplified by polymerase chain reaction (PCR) using the set of
primers ITS1 (5′-CTT GGT CAT TTA GAG GAA GTA A-3′) and ITS4 (5′-TCC TCC GCT
TAT TGA TATGC-3′) [21]. Amplification reactions were performed in a final volume of 25 μL
containing 50 ng of genomic DNA (template), 20 mM Tris–HCl, pH 8.4, 50 mM KCl, 3.0 mM
MgCl2, 200 μM of each dNTP (Thermo Scientific, Lithuania, EU), 20.0 pmoles of each
primer, and 1.0 U of Taq DNA Polymerase (Thermo Scientific, Lithuania, EU). The PCR
cycles were composed by denaturing for 1 min at 94 °C, annealing for 1 min at 52 °C, and
extension for 2 min at 72 °C for a total of 30 cycles, followed by a final extension at 72 °C for
8 min. The amplified fragments were purified using Wizard® SV Gel and PCR Clean-Up
System (Promega, Madison, WI, US). The sequencing reactions were carried out with primers
ITS1 and ITS4 using the ABI PRISM BigdyeTM terminator cycle sequencing kit (Applied
Biosystems, Foster City, CA, USA) following the protocol supplied by the manufacturer. The
fluorescently labeled fragments were purified by ethanol precipitation, suspended in water, and
subjected to electrophoresis in an ABI 3730 sequencer (Applied Biosystems, Foster City, CA,
USA). The partial sequences were used to generate a consensus sequence. The obtained
consensus sequence was compared to sequences within the NCBI database (http://www.
ncbi.nlm.nih.gov/) using the Basic Local Alignment Search Tool (BLAST).

Ethanol Production by the Isolated Yeast

The selected yeast was inoculated on Agar YEPD and incubated at 30 °C for 48 h. Inoculum
preparation was performed in 500-mL Erlenmeyer flask with a medium volume of 300 mL of
YEPD. The growth was carried out at 30 °C and 150 rpm on an orbital shaker for 24 h. After
2766 Appl Biochem Biotechnol (2014) 174:2762–2776

that, cells were centrifuged at 10,000g for 10 min to obtain the initial biomass used in
fermentation assays (5.00 g L−1 ±1 g L−1).
The effect of temperature on ethanol production was studied using a synthetic medium,
composed of 45 g L−1 glucose, 45 g L−1 fructose, 2.5 g L−1 (NH4)2SO4, 0.5 g L−1 KH2PO4,
0.65 g L−1 MgSO4·7H2O, and 0.65 g L−1 ZnSO4, with an initial medium pH of 4.5 adjusted
with 1 M HCl or 1 M NaOH. The concentration of glucose and fructose in the synthetic
medium was the same to those found in the CAJ. Fermentations were conducted in 500-mL
Erlenmeyer flasks with 250-mL medium at different temperatures (28, 30, 32, 34, 36, and
38 °C) and 150 rpm in an orbital shaker (Tecnal TE-420, Piracicaba, SP, Brazil).
CAJ and MCAB-OH were fermented at 500-mL Erlenmeyer flasks, containing 250-mL
medium at 32 °C and 150 rpm in an orbital shaker (Tecnal TE-420, Piracicaba, SP, Brazil).
When the influence of the initial pH medium was evaluated, CAJ was used and the initial pH
values studied were 3.0, 4.5, 5.5, and 6.3, adjusted with 1 M HCl and 1 M NaOH.
During fermentation, samples were withdrawn at predefined intervals and analyzed for cell
growth, pH, substrate concentration, and ethanol concentration. All assays were conducted in
triplicate.

Analytical Methods

Cell concentration was determined by a turbidimetric method at 660 nm in a Spectronic 20

Genesys spectrophotometer. The concentration (g L−1) was calculated from the calibration
curve constructed for the selected yeast. The concentrations of sugars (glucose, fructose, and
xylose) and ethanol were measured by high-performance liquid chromatography (HPLC;
Waters, Milford, MA, USA) equipped with refractive index detector (Waters 2414) and an
Aminex column HPX-87H (Bio-Rad, Hercules, CA, USA). Five millimoles per liter sulfuric
acid was used as the mobile phase at a flow rate of 0.5 mL min−1, and the analysis was
conducted at 65 °C. The injection volume of samples was 20 μL. The samples were identified
by comparing retention times to the retention time of the standard samples.

Fermentation Parameters

Fermentation parameters, such as maximum specific growth rate (μmax), conversion of

substrate to product (YP/S), conversion of substrate to cells (YX/S), the yield of product based
on cell growth (YP/X), ethanol volumetric productivity (QP), sugar conversion (θS), and
efficiency of sugar conversion to ethanol (η), were estimated at the end of the fermentation
process, as defined by Pacheco et al. [22], using Eqs. 1–6, respectively.

lnðX − X i Þ
μmax h−1 ¼ ð1Þ
  t
P
Y P=S =g ¼
g
ð2Þ
S −S
  X i−X
i
Y X =S =g ¼
g
ð3Þ
S i −S
  P
Y P=X g =g ¼ ð4Þ
X −X i

P
QP g:L−1 :h−1 ¼ ð5Þ
t
P
θS ð%Þ ¼ ð6Þ
0; 511:ðS i − S Þ
Appl Biochem Biotechnol (2014) 174:2762–2776 2767

P
η ð%Þ ¼ ð7Þ
0:511:ðS i − S Þ

where X is the cell concentration (g L−1), Xi is the initial cell concentration (g L−1), S is the
glucose concentration (g L−1), Si is the initial glucose concentration (g L−1), P is the ethanol
concentration (g L−1), and μmax is the maximum specific growth rate. In Eq. 7, the value 0.511
corresponds at YP/S theoretical.
The yields (YP/S) of first- and second-generation ethanol production were determined using
the same equation (Eq. 2). These yields were based on the concentration of carbohydrate
present in the medium (synthetic medium, CAJ, or CAB hydrolysate).
Ethanol volume-to-volume percentage (alc/vol, %) was determined as the ratio of the
volume of alcohol per volume of broth at the end of the assay, multiplied by 100. For
comparative analysis between the fermentative processes, fermentation parameters were
subjected to the ANOVA statistical test performed with Origin Pro 8.0 (Microcal Origin Pro
8.0) software to evaluate the deviation of the variances of a group of the experimental data in
order to predict, with a confidence level of 95 %, if the population of experimental data has any
significant difference.

Results and Discussion

Isolation and Identification of an Ethanol-Producing Yeast from CAJ

During the fermentation of CAJ, an increase in turbidity after 24 h was observed suggesting
microbial growth. The cultivation of the fermented CAJ in Sabouraud agar led to the isolation
of a predominant translucent colony of yeast coded, GPBIO03. The molecular identification of
the rRNA operon encompassing the 5.8S rRNA gene and the flanking internal transcribed
spacers ITS1 and ITS2 was determined, and it had a length of 665 bp. Similarity searches on
public nucleotide databases using that sequence revealed 100 % identity with Hanseniaspora
opuntiae and Hanseniaspora uvarum. Therefore, the yeast strain was classified as
Hanseniaspora sp. GPBIO03. The sequence was deposited in GenBank under the accession
number KF791566.
Several authors have used different Hanseniaspora strains in the fermentation processes.
Escalante et al. [23] evaluated the fermentative activity of H. uvarum using grape juice to
produce fermented beverages. Andorra et al. [13] tested Hanseniaspora guilliermondii for
ethanol production. Pina et al. [14] studied the tolerance of non-Saccharomyces strains to
produce ethanol, in which two belonged to the genus Hanseniaspora. Thus, yeast strains
belonging to the genus Hanseniaspora have been used in various fermentation processes.
Therefore, the yeast isolated from CAJ and identified as Hanseniaspora sp. GPBIO03 was
evaluated for the production of ethanol (first- and second-generation). First of all, the effects of
temperature and pH on first-generation ethanol production were determined.

Influence of Temperature on Ethanol Production

Figure 1 shows the influence of temperature on ethanol production by Hanseniaspora sp.

GPBIO03 using a synthetic medium. It is possible to see that from 28 to 36 °C, no significant
difference is observed at ethanol concentration during the assay. The same behavior was
observed for glucose and fructose consumption, as well as biomass production. On the other
hand, when the fermentation was conducted at 38 °C, lower values of ethanol and biomass
 2768 Appl Biochem Biotechnol (2014) 174:2762–2776

10 50
9 (A) 45 (B)

Glucose Concentration (g L )
8 40

-1
Cell Concentration (g.L )
-1

7 35
6 30
5 25
4 20
3 15
2 10
1 5
0 0
0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18
Time (h) Time (h)

50 50
45 (C) 45 (D)
Fructose Concentration (g.L )

Ethanol Concentration (g.L )

-1

-1
40 40
35 35
30 30
25 25
20 20
15 15
10 10
5 5
0 0
0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18
Time (h) Time (h)

Fig. 1 Effect of temperature on substrate (glucose and fructose) consumption, biomass, and ethanol production
by Hanseniaspora sp. GPBio03 at 150 rpm using a synthetic medium: 28 °C (inverted triangle), 30 °C (triangle),
34 °C (diamond), 36 °C (circle), and 38 °C (square)

were obtained. Table 1 resumes the effect of temperature on the kinetic parameters at the range
evaluated. No significant (p<0.05) difference was observed in the conversion of the substrate
into product (YP/S) in all tested temperatures. Likewise, efficiency was not significantly
affected by the temperature. The yield of product (YP/S) was similar at temperatures of 28,

Table 1 Influence of the temperature on ethanol production by Hanseniaspora sp. GPBIO03 using synthetic
medium

Parameter Temperature

28 °C 30 °C 32 °C 34 °C 36 °C 38 °C

μmax (h−1) 0.050±0.00 0.054±0.00 0.064±0.00 0.063±0.00 0.069±0.00 0.054±0.00

YP/S (g g−1) 0.44±0.00 0.44±0.02 0.44±0.02 0.43±0.00 0.45±0.02 0.43±0.01
YP/X (g g−1) 11.88±1.93 10.99±0.19 11.19±0.43 11.43±0.12 10.87±2.47 9.34±0.75
YX/S (g g−1) 0.03±0.01 0.03±0.01 0.04±0.00 0.04±0.00 0.04±0.00 0.05±0.01
Pmax (g L−1) 37.98±0.89 37.14±1.02 37.07±0.37 36.92±0.20 37.60±0.52 32.40±0.57
alc/vol (%) 4.81±0.11 4.71±0.13 4.69±0.03 4.67±0.12 4.80±0.14 4.11±0.08
QP (g L−1 h−1)a 2.37±0.06 2.32±0.06 2.31±0.02 2.24±0.12 2.35±0.03 1.95±0.13
θS (%) 99.53±0.17 99.72±0.00 97.77±1.35 99.12±0.43 98.57±0.56 83.66±1.76
η (%) 86.56±0.79 86.59±4.41 86.98±3.69 84.15±0.68 86.11±1.20 84.92±2.06
a
Productivity determined at 12 h of fermentation
Appl Biochem Biotechnol (2014) 174:2762–2776 2769

30, 32, 34, and 36 °C but was significantly different at 38 °C, reaching its lowest value
(9.34 g g−1 ±0.75 g g−1). The substrate yield on cells (YX/S) was similar in the range of 28 to
36 °C, reaching its highest value at the temperature of 38 °C. On the other hand, lower ethanol
production, productivity, and yield of product based on cells (YP/X) were achieved at this
temperature. Therefore, the most suitable range for the operating process is at 28 to 36 °C.
The yeast consumed all the glucose and fructose (total sugar) during 16 to 18 h when grown
in the temperature range from 28 to 36 °C, but when cultured at 38 °C, about 5 g L−1 of total
reducing sugars was still present in the fermentation medium (Fig. 1b, c). The cell growth
profile (Fig. 1a) was similar at all tested temperatures, reaching an average of 18 g L−1 of cells
in the range of 28 °C to 36 °C and a lowest concentration at 38 °C (15 g L−1 cells). The higher
ethanol concentrations obtained were similar in the assays conducted at 28, 30, 32, 34, and
36 °C and were approximately 37 g L−1 of ethanol. However, there was a decrease in the
maximum production at 38 °C, reaching 32 g L−1 ethanol. These results indicate that the strain
GPBIO03 is able to metabolize glucose and fructose for the production of ethanol at temper-
atures lower than 38 °C.
The results obtained in this study were different from those obtained by Torija et al. [24],
which evaluated the effect of temperature on the kinetics of Saccharomyces cerevisiae, and
stated that alcoholic fermentation is affected by temperature, with the highest yield at lower
temperatures of 15–20 °C. In their study, when the bioreactor temperature was in the range of
25–30 °C, the initial fermentation rate was higher, and above 35 °C, a decrease in cell viability
occurs, whereas in the present study, loss of viability occurred only when the yeast
Hanseniaspora sp. GPBIO03 was grown at 38 °C.
Several studies with Hanseniaspora strains using grape or apple juice for alcoholic
fermentation were conducted at temperatures between 25 and 28 °C [13, 17, 23, 25, 26].
The results of the fermentation parameters obtained in this work are higher than those
obtained by Escalante et al. [23]. These authors used apple juice as culture medium with an
initial substrate concentration of 127.2 g L−1 at 28 °C, which obtained the following param-
eters: specific cell growth rate, YP/S, and YX/S of 0.05 h−1, 0.15 g g−1, and 0.062 g g−1,
respectively, after 22 h of fermentation.
Based on the obtained results, the average temperature of 32 °C was selected to conduct the
next steps, using synthetic media, CAJ, or MCAB-OH.

First- and Second-Generation Ethanol Production by Hanseniaspora sp. GPBIO03

Figure 2a, b, c shows the profiles of biomass, substrate, and ethanol obtained of the fermen-
tation with Hanseniaspora sp. GPBIO03 using synthetic medium, CAJ, and MCAB-OH,
respectively.
The profile of cell growth was similar in all medium evaluated, being produced an average
of 10 g L−1 cells. In general, Hanseniaspora, which is usually present in the early stages of
wine production by natural fermentation of grape juice, shows limited growth time, due to its
low tolerance to high concentrations of ethanol. Species such as H. guilliermondii and
H. uvarum, for example, grow between 4 and 6 days of fermentation and then die largely as
a result of increased ethanol concentration produced by strains of S. cerevisiae, also present in
the broth, which has greater tolerance to alcohol. Generally, species of Candida, Pichia,
Metschnikowia, and Hanseniaspora found in grape juice are not tolerant to higher concentra-
tions of ethanol, superior to 4–7 % [17, 27–29].
On the other hand, Pina et al. [14] showed that H. guilliermondii and S. cerevisiae have
similar tolerance to ethanol, presenting cell growth in an ethanol concentration of 25 % (v/v),
and this yeast is more tolerant than other apiculate yeasts, for example, H. uvarum. In the
2770 Appl Biochem Biotechnol (2014) 174:2762–2776

present work, the cell growth of Hanseniaspora sp. GPBIO03 was observed until the end of
fermentation in all evaluated media, when the concentration of ethanol was 38 and 40 g L−1 for
the synthetic media and CAJ (Fig. 2a, b, respectively) and 21.56 g L−1 ethanol for MCAB-OH
(Fig. 2c).
The yeast consumed all the glucose and fructose (total sugars) in the period 16–18 h when
cultivated in synthetic medium (Fig. 2a), but in the CAJ medium at 10 h of fermentation, total
reducing sugar concentration was about 10 g L−1 (Fig. 2b). The maximum ethanol production
was similar when using synthetic medium or CAJ medium, but the highest concentration was
obtained at CAJ. These results indicate that the yeast Hanseniaspora sp. GPBIO03 is able to
metabolize glucose and fructose to produce first-generation ethanol.
When grown in MCAB-OH (Fig. 2c), the yeast Hanseniaspora sp. GPBIO03 produced the
highest ethanol concentration (24.37 g L−1 ±0.68 g L−1) at 5 h, and all glucose was metabo-
lized by the yeast during this period. These results indicate that the yeast isolated is also able to
produce second-generation ethanol.
Table 2 shows a comparison of the ethanol production by the yeast in the three fermentation
media evaluated. The highest maximum specific cell growth rate (0.06 h−1 ±0.01 h−1), con-
version of substrate to product (0.48 g g−1 ±0.02 g g−1), productivity of ethanol (4.40 g L−1 h−1
±0.72 g L−1 h−1), and efficiency (95.96 %±1.64 %) were obtained using CAJ as a carbon
source, and thereby the juice is the best substrate for ethanol production by this strain. These
results are probably due to the superior nutritional value of CAJ, which contains amino acids,
vitamins, and minerals, according to Rocha et al. [30].
When MCAB-OH was used, ethanol productivity was 4.87 g L−1 h−1, with an efficiency of
80.23 % and YP/S of 0.41 g g−1 (Table 2), which was equivalent to 2 g of ethanol per 100 g of

100 50 100 50

(A) Synthetic Medium (B) Cashew Apple Juice

Cells and Ethanol Concentration (g.L )

90 45
-1

45

Cells and Ethanol concentration (g.L )

80
Sugars concentration (g.L )

40 80 40
-1

Sugars concentration (g.L )

-1

35 70 35
60
30 60 30

25 50 25
40
20 40 20

15 30 15
20
10 20 10

0 5 10 5
-1

0 0 0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 0 1 2 3 4 5 6 7 8 9 10 11

Time (h) Time (h)

60 50
(C) MCAB-OH Medium
Cells and Ethanol concentration (g.L )
-1

45
Glucose concentration (g.L )

50
-1

40

35
40
30

30 25

20
20
15

10
10
5

0 0
0 1 2 3 4 5 6 7 8 9
Time (h)

Fig. 2 Ethanol production by Hanseniaspora sp. GPBio03 at 32 °C and 150 rpm: synthetic medium (a), CAJ
(b), and MCAB-OH (c). The experimental data are cell concentration (g L−1) (square), ethanol concentration
(g L−1) (circle), and sugar (glucose + fructose) concentration (g L−1) (triangle). Line of tendency (—)
Appl Biochem Biotechnol (2014) 174:2762–2776 2771

Table 2 Parameters of ethanol production by Hanseniaspora sp. GPBIO03 at 32 °C using synthetic medium,
cashew apple juice (CAJ), and cashew apple bagasse hydrolysate (MCAB-OH) as carbon sources

Parameter Fermentation medium

Synthetic CAJ MCAB-OH

μmax (h−1) 0.05±0.00 0.10±0.00 0.08±0.00

YP/S (g g−1) 0.44±0.02 0.48±0.02 0.41±0.01
YP/X (g g−1) 11.19±0.43 9.16±0.44 6.86±0.01
YX/S (g g−1) 0.04±0.00 0.06±0.00 0.08±0.00
Pmax (g L−1) 38.00±0.20 41.30±1.00 24.37±0.68
alc/vol (%) 4.69±0.03 5.07±0.19 3.04±0.08
QP (g L−1 h−1) 2.31±0.02a 4.40±0.72a 4.87±0.03b
θS (%) 97.77±1.35 95.22±0.16 100±0.00
η (%) 86.98±3.69 95.96±1.64 80.23±0.02
a
Productivity determined at 12 h of bioprocess
b
Productivity determined at 5 h of bioprocess

initial raw material. It is noteworthy that the enzymatic hydrolysate of CAB (MCAB-OH) has
a low concentration of substrate compared to synthetic and CAJ media. However, these results
indicate that yeast can also be applied in the production of second-generation ethanol, which
was the objective of this work.
Several studies have investigated the production of second-generation ethanol using differ-
ent lignocellulosic materials such as CAB [7, 9, 31], sugarcane bagasse [32], wheat straw [33],
and corn stover [34] using the yeast S. cerevisiae, and the results of YP/S are similar or lower
than to those obtained in the present work.
Figure 3 shows a flowchart and the mass balance of the process to represent the integration of
first- and second-generation ethanol production using cashew apple as feedstock. The cashew
apple juice (CAJ) was used as carbon source and it contains 90 g L−1 of carbohydrate (glucose and
fructose). The results obtained for this process were 41.3 g L−1 ethanol, with a yield and efficiency
of 0.48 g g−1 and 96 %, respectively. Considering 100-g cashew apple bagasse as basis, a
schematic diagram for second-generation ethanol production was carried out. Initially, CAB
was pretreated using H2SO4 (0.6 mol L−1), solid concentration of 30 % (w/v) at 121 °C for
15 min. The solid and liquid fractions were separated, and 61-g solid fraction was recovered and
nominated of CAB hydrolysate (CAB-H). Afterward, the solid fraction (CAB-H) was treated
using NaOH (4 % w/v), at a solid concentration of 7.5 % (w/v) at 121 °C for 30 min. It can be seen
that 9.3 g of the solid fraction is recovered. Next, enzymatic hydrolysis was conducted using
CAB-OH, and the hydrolysate was separated from the remaining solid fraction prior to ethanol
production using Hanseniaspora sp. In this step, the sugar yield was equivalent to 5 g of glucose
per 100 g of initial raw material, thus reaching 85.6 g L−1 of concentration in the liquid
hydrolysate, which was adjusted for 50 g L−1 with distillated water. Finally, the yield of ethanol
was 0.41 g g−1, which represents approximately 24.4 g L−1 ethanol, 80 % of efficiency that could
be theoretically obtained and a yield of 20 mg ethanol g−1 CAB (20 kg t−1 CAB).

Influence of Initial pH of CAJ on Ethanol Production

The profile of cell growth, consumption of sugars (glucose and fructose), and ethanol
production was similar at all values of pH evaluated, see Fig. 4. However, the highest ethanol
2772 Appl Biochem Biotechnol (2014) 174:2762–2776

production was achieved when initial pH was 3.0 (approximately 44 g L−1). At pH 4.4, a
concentration of ethanol of 42 g L−1 was produced with a lower consumption of substrate
(Fig. 4b, c). Cell growth was lower in the initial pH 3.0 in which 7.5 g L−1 of biomass
concentration was obtained (Fig. 4a). Figure 5 shows the evolution of pH throughout the
fermentation with Hanseniaspora sp. GPBIO03 in the CAJ medium. In the media with
initial pH 5.5 or 6.3, there was a decrease on the pH value at the beginning of the
fermentation, which remained constant and equal to 4.5 at the end of each process.
When the initial pH of the CAJ medium was adjusted to 3.0 or 4.5, it remained
constant, thus demonstrating the buffering capacity of the CAJ. This capacity was also
verified by Assad et al. [35], which studied the buffering capacity of 15 fruit juices
by adding 0.1 N NaOH to 10 mL of a sample and found that 2.5 mL of the
concentrated NaOH solution is needed to raise the cashew juice pH to 7.0.

Fig. 3 Schematic flowchart and mass balance of the process to represent the integration of first- and second-
generation ethanol production using cashew apple as feedstock
Appl Biochem Biotechnol (2014) 174:2762–2776 2773

12 50
11 45 (B)
10

Glucose Concentration (g.L-1)

40
Cells Concentration (g.L )

9
-1

35
8
30
7
6 25
5 20
4 15
3
10
2
5
1
0 0
0 1 2 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 6 7 8 9 10 11
Tempo (h) Time (h)

50 50
45 (C) 45
(D)
Fructose Concentration (g.L )
-1

Ethanol Concentration (g.L )

40
-1
40
35 35
30 30
25 25
20 20
15 15
10 10
5 5

0 0
0 1 2 3 4 5 6 7 8 9 10 11 0 1 2 3 4 5 6 7 8 9 10 11
Time (h) Time (h)

Fig. 4 Effect of initial pH on substrate (glucose and fructose) consumption, biomass, and ethanol production by
Hanseniaspora sp. GPBio03 grown on cashew apple juice at 32 °C and 150 rpm: 3.0 (square), 4.5 (circle), 5.5
(triangle), and 6.3 (inverted triangle)

Fig. 5 pH values along time during ethanol production by Hanseniaspora sp. GPBIO03 using cashew apple juice
(CAJ) at 32 °C and 150 rpm. Initial pH at 3.0 (square), 4.5 (circle), 5.5 (triangle), and 6.3 (inverted triangle)
2774 Appl Biochem Biotechnol (2014) 174:2762–2776

The effect of pH on the maximum specific growth rate and in the YP/X, YX/S, and YP/S yields,
productivity, efficiency, and maximum production of ethanol was also evaluated, with the
results shown in Table 3.
The pH did not influence the yield of substrate to product (YP/S), since the values were
similar with a confidence level of 95 %. The cell yield in product (YP/X) decreased with
increasing initial pH, reaching the maximum value (12.43 g g−1) at pH 6.3 and the minimum
value (6.06 g g−1) at pH 3.0. The lowest value of YX/S was observed at pH 3.0 (0.04 g g−1),
demonstrating that at this pH (3.0), the cells were subjected to metabolic stress [36].
The yields, the maximum ethanol production, and efficiency were similar at all pH values,
demonstrating that the yeast was adapted to the environment in all conditions of pH.
Pina et al. [14], studying the ethanol tolerance of five non-Saccharomyces yeast strains
(among which two were from the genus Hanseniaspora), used an initial pH of 3.8, 4.5, or 3.5
in their trials, and the results obtained demonstrated growth of Hanseniaspora strains at pH 3.5
to 4.5. These data corroborate the results obtained in this study.
Dorta [37] cited that pH 4.5 was sufficient to minimize the harmful effects of
sulfite and ethanol on the cells. It is noteworthy that the fermentations conducted in
more acidic media resulted in higher yields of ethanol due to low production of
glycerol. This condition also helps to control infection as it reduces the growth of
contaminating bacteria.
Therefore, the range of pH 3.0 to 5.5 is the more suitable to the process and is mostly used
for yeasts of the genus Hanseniaspora, leading to a higher yield of ethanol, but also enables
better control of infection and reduces the possibility of contamination of the process on an
industrial scale where the conditions of perfect sterilization are more difficult to maintain.
According to Tables 1, 2, and 3, the maximum ethanol volume-to-volume percentage (alc/
vol) is between 4 and 6 %. These results are similar to those of other authors [26] that studied
the properties of oenological yeast non-Saccharomyces on fermentation of grape and using
initial concentration of 270 g L−1 of total sugars and stated that the yeast produced 4–6 %
ethanol, with a maximum of 6.07 % achieved by H. uvarum. However, the results obtained
from the fermentation using Hanseniaspora sp. GPBIO03 reached the same range of ethanol
production with a threefold lower concentration of total sugars, when compared to the
experiments presented by Ciani et al. [26].

Table 3 Influence of pH on the ethanol production by Hanseniaspora sp. GPBIO03 at 32 °C using cashew
apple juice (CAJ) as carbon source

Parameter pH

3.0 4.5 5.5 6.3

μmax (h−1) 0.104±0.00 0.100±0.00 0.106±0.00 0.140±0.01

YP/S (g g−1) 0.48±0.02 0.48±0.01 0.49±0.01 0.48±0.00
YP/X (g g−1) 12.43±0.25 9.16±0.12 8.45±0.15 6.06±0.20
YX/S (g g−1) 0.04±0.01 0.06±0.00 0.05±0.01 0.07±0.01
Pmax (g L−1) 43.26±0.89 41.05±1.10 42.10±1.11 40.03±0.59
v/v (%) 5.48±0.11 5.07±0.19 5.34±0.14 5.20±0.07
QP (g L−1 h−1)a 4.33±0.09 4.10±0.05 4.17±0.11 4.10±0.06
θS (%) 93.74±1.74 92.32±1.48 94.00±0.82 93.80±0.47
η (%) 94.83±3.05 93.23±3.64 95.89±2.77 93.71±1.21
a
Productivity at 10 h of bioprocess
Appl Biochem Biotechnol (2014) 174:2762–2776 2775

Conclusions

The yeast Hanseniaspora sp. GPBIO03, isolated from CAJ, has shown to be a promising
strain for first- and second-generation ethanol production using CAJ and enzymatic hydroly-
sate of CAB as carbon sources. The optimum conditions of temperature and pH of the process
range from 28 to 36 °C and pH 3.0 to 5.5.

Acknowledgments The authors would like to thank the Brazilian research-funding agencies CNPq and
CAPES.

References

1. Dias, M. O. S., Modesto, M., Ensinas, A. V., Nebra, S. A., Maciel Filho, R., & Rossell, C. E. V. (2011).
Energy, 36, 3691–3703.
2. Medeiros, V. (2011). Revista Petrobrás.
3. Ojeda, K., Ávila, O., Suárez, J., & Kafarov, V. (2011). Chemical Engineering Research and Design, 89, 270–
279.
4. Alvira, P., Tomás-Pejó, E., Ballesteros, M., & Negro, M. J. (2010). Bioresource Technology, 101, 4851–
4861.
5. Machado, C. M. M. (2010) Avaliable from: http://www.embrapa.br/imprensa/artigos/2009/producao-de-
biocombustiveis-pormicrorganismos. Accessed 08 Aug 2012.
6. Pinheiro, A. D. T., Rocha, M. V. P., Macedo, G. R., & Gonçalves, L. R. B. (2008). Applied Biochemistry and
Biotechnology, 148, 227–234.
7. Rocha, M. V. P., Rodrigues, T. H. S., Macedo, G. R., & Goncalves, L. R. B. (2009). Applied Biochemistry
and Biotechnology, 155, 104–114.
8. Neelakandan, T., & Usharani, G. (2009). America-Eurasian Journal of Scientific Research, 4(2), 85–88.
9. Rodrigues, T. H. S., Rocha, M. V. P., Macedo, G. R., & Gonçalves, L. R. B. (2011). Applied Biochemistry
and Biotechnology, 164, 929–943.
10. Deenanath, E. V., Rumbold, K., & Iyuke, S. (2013). ISRN Renewable Energy, 1–11.
11. SÍNTESE - Conjuntural da safra de castanha-de-caju 2010/2011. Companhia Nacional de Abastecimento –
CONAB. Rio Grande do Norte, 2010.
12. Correia, J. A. C., Marques Junior, J. E., Gonçalves, L. R. B., & Rocha, M. V. P. (2013). Bioresource
Technology, 139, 249–256.
13. Andorra, I., Monteiro, M., Esteve-Zarzoso, B., Albergaria, H., & Mas, A. (2011). Food Microbiology, 28,
1483–1491.
14. Pina, C., Santos, C., Couto, J. A., & Hogg, T. (2004). Food Microbiology, 21, 439–447.
15. Medina, K., Boido, E., Fariña, L., Gioia, O., Gomez, M. E., Barquet, M., Gaggero, C., Dellacassa, E., &
Carrau, F. (2013). Food Chemistry, 141, 2513–2521.
16. Tao, N., Gao, Y., & Liu, Y. (2011). Brazilian Journal of Microbiology, 42(2), 668–675.
17. Moreira, N., Pina, C., Mendes, F., Couto, J. A., Hogg, T., & Vasconcelos, I. (2011). Food Control, 22, 662–
667.
18. Rivera, E. C., Costa, A. C., Atala, D. I. P., Maugeri, F., Maciel, M. R. W., & Filho, R. (2006). Process
Biochemistry, 41, 1682–1687.
19. Akin, H., Brandam, C., & Meyer, X. (2008). Chemical Engineering and Processing: Process Intensification,
47(11), 1986–1993.
20. Warner, S. A. J. (1996). Genomic DNA isolation and lambda library construction in plant gene isolation:
principles and practice. In G. D. Foster & D. Twell (Ed.) (pp. 51–73). New York: Wiley.
21. White, T. J., Bruns, T., Lee, S. B., & Taylor, J. W. (1990). Amplification and direct sequencing of fungal
ribosomal RNA genes for phylogenetics. In M. A. Innis, D. H. Gelfand, J. J. Sninky, & T. J. White (Eds.),
PCR protocols: a guide to methods and applications (pp. 315–322). New York: Academic.
22. Pacheco, A. M., Gondim, D. R., & Gonçalves, L. R. B. (2010). Applied Biochemistry and Biotechnology,
161, 209–217.
23. Escalante, E. W., Rychtera, M., Melzoch, K., Sakoda, B. H., Polo, E. Q., Cervantes, Z. L., Casavilca, V. S., &
Quilca, G. C. (2011). Revista de la Sociedad Venezolana de Microbiología, 31, 57–63.
24. Torija, M. J., Rozès, N., Poblet, M., Guillamón, J. M., & Mas, A. (2003). International Journal of Food
Microbiology, 80(1), 47–53.
2776 Appl Biochem Biotechnol (2014) 174:2762–2776

25. Ciani, M., & Picciotti, G. (1995). Biotechnology Letters, 17, 1247–1250.
26. Ciani, M., & Maccarelli, F. (1998). World Journal of Microbiology and Biotechnology, 14, 199–203.
27. Fleet, G. H. (2003). International Journal of Food Microbiology, 86, 11–22.
28. Jolly, N., Augustyn, O., & Pretorius, I. (2006). South African Journal of Enology and Viticulture, 27, 15–39.
29. Moreira, N., Mendes, F., Guedes de Pinho, P., Hogg, T., & Vasconcelos, I. (2008). International Journal of
Food Microbiology, 124, 231–238.
30. Rocha, M. V. P., Gomes, R. V., Melo, V. M. M., & Gonçalves, L. R. B. (2009). Applied Biochemistry and
Biotechnology, 155, 63–75.
31. Rocha, M. V. P., Rodrigues, T. H. S., Albuquerque, T. L., Gonçalves, L. R. B., & Macedo, G. R. (2014).
Chemical Engineering Journal, 243, 234–243.
32. Rocha, G. J. M., Martin, C., Soares, I. B., Maior, M. A. S., Baudel, H. M., & Abreu, C. A. M. (2011).
Biomass and Bioenergy, 35, 663–670.
33. Govumoni, S. P., Koti, S., Kothagouni, Y. S., Venkateshwar, S., & Linga, V. R. (2013). Carbohydrate
Polymers, 91(2), 646–650.
34. Saha, B. C., Yoshida, T., Cotta, M. A., & Sonomoto, K. (2013). Industrial Crops and Products, 44, 367–372.
35. Assad, A. M. E., Netto, J. D. M., Losso, E. M., Torres, M. F., & Brancher, J. A. (2010). Revista Sul-
Brasileira de Odontologia, 7(3), 281–286.
36. Moser, A. (1985). Kinectis of batch fermentation. In H. J. Rehm, G. Reed, & H. Brauer (Eds.),
Biotechnology—a comprehensive treatise in 8 volumes (Vol. 2, pp. 243–283). Weinheim: V.C.H.
Verlagsgesellschaft.
37. Dorta, C., Oliva-Neto, P., Abreu-Neto, M. S., Nicolau-Junior, N., & Nagashima, A. I. (2006). World Journal
of Microbiology and Biotechnology, 22(2), 177–182.