Bioprocess and Biosystems Engineering

https://doi.org/10.1007/s00449-019-02136-3

RESEARCH PAPER

Ethanol production from water hyacinth (Eichhornia crassipes)

hydrolysate by hyper‑thermal acid hydrolysis, enzymatic
saccharification and yeasts adapted to high concentration of xylose
InYung Sunwoo1 · Jeong Eun Kwon1 · Trung Hau Nguyen1 · Gwi‑Tack Jeong1 · Sung‑Koo Kim1

Received: 17 February 2019 / Accepted: 24 April 2019

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Water hyacinth (Eichhornia crassipes) was used as a feedstock for ethanol production. The optimal hyper-thermal (HT) acid
hydrolysis conditions were 8% (w/v) slurry content, 200 mM ­H2SO4, at 160 °C for 20 min and enzymatic saccharification for
48 h using an enzyme mixture of 20 units/mL Viscozyme L and Cellic C Tec2. After pretreatment, 48.2 g/L monosaccharides
were obtained. Fermentation was conducted with wild and adapted Saccharomyces cerevisiae, Pichia stipitis and Candida
lusitaniae. Wild-type S. cerevisiae, P. stipitis, and C. lusitaniae produced 15.3, 19.5 and 22.7 g/L of ethanol, respectively.
Adaptive evolution was carried out on 6% (w/v) xylose. S. cerevisiae, P. sipitis and C. lusitaniae adapted to xylose produced
15.3, 21.4 and 23.9 g/L of ethanol with YEtOH of 0.32, 0.44 and 0.49, respectively. These results indicate that water hyacinth
has potential as a feed stock for ethanol.

Keywords  Adaptive evolution · Enzymatic saccharification · Ethanol production · Fermentation · Hyper-thermal acid
hydrolysis · Water hyacinth (Eichhornia crassipes)

Introduction water hyacinth is a promising raw material for ethanol pro-

Global crude oil production is predicted to decline due to a
shortage of fossil fuels. Greenhouse gases contain exceed-
ingly high levels of ­CO2, reaching approximately 450 ppm.
The need to develop alternative and sustainable energy
sources has already prompted many research projects world-
wide [1]. The use of non-food biomass for second-generation
ethanol production even now lacks economic feasibility [2].
The water hyacinth (Eichhornia crassipes) covers vast
areas of the Vam Co Dong River, Vietnam, blocks rivers and
channels, eliminates of other aquatic plants and restricts the
light and air in the underwater environment [3]. However,
cleaning and disposing of water hyacinth is expensive and
requires high manpower. Thus, solutions are required to effi-
ciently handle water hyacinth. Recent studies reported that
InYung Sunwoo and Jeong Eun Kwon have equally contributed as
co-first authors.
* Sung‑Koo Kim
skkim@pknu.ac.kr
1
Department of Biotechnology, Pukyong National University,
Busan 48513, South Korea
duction due to fast growth rate and low lignin contents [4, 5].
One requirement for the pretreatment of biomass is the
hydrolysis of polysaccharides to monosaccharides without
the formation of inhibitors, such as 5-hydroxymethylfurfural
(HMF), levulinic acid, acetic acid and formic acid. These
inhibitors can retard yeast growth and reduce ethanol pro-
ductivity during fermentation [6]. Therefore, hyper-thermal
(HT) acid hydrolysis is conducted to minimize the degrada-
tion of monosaccharides into inhibitory byproducts [7, 8].
The fermentation organism should be capable of ferment-
ing all monosaccharides present in the hydrolysate. Sac-
charomyces cerevisiae is the most commonly used ethanol
producer; however, wild-type S. cerevisiae cannot ferment
pentose, which comprises up to 40% of water hyacinth con-
tent [9]. Among xylose-fermenting yeasts, Pichia stipitis
and Candida lusitaniae have demonstrated the ability to
ferment xylose to ethanol [10, 11]. However, these yeasts
prefer glucose and exhibit limited assimilation of other
monosaccharides, such as galactose or xylose from a mixed
sugar medium. Therefore, it is desirable to enhance galactose
and/or xylose consumption in P. stipitis and C. lusitaniae
through adaptive evolution.

13
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In this study, bioethanol was produced from the water
hyacinth E. crassipes, with HT acid hydrolysis as a pretreat-
ment for glucose production. Various enzymes were then
used for enzymatic saccharification to produce more glucose
and xylose. Fermentation to xylose was conducted using
wild and yeasts adapted to determine optimal fermentation
conditions.
Materials and methods
Composition of water hyacinth
Water hyacinths were obtained from the Vam Co Dong
River, Tay Ninh, Vietnam, dried to a constant weight at
60 °C, ground with a roller mill, and sieved with a 200-
mesh sieve prior to pretreatment. Composition analysis was
performed by the Feed and Food Nutrition Research Center
at Pukyong National University, South Korea, according to
the Association of Official Analytical Chemists (AOAC)
method [12].
Water hyacinth composition was 38.7% carbohydrate,
12.5% crude protein, 0.36% crude lipid, 19.4% crude ash
and 29.0% fiber. The total carbohydrate content was 67.7%.
HT acid hydrolysis and enzymatic saccharification
The HT acid hydrolysis and enzymatic saccharification pre-
treatment processes were optimized. HT acid hydrolysis was
conducted sequentially, focusing on the optimal effects of
four factors: ­H2SO4 concentration [100–400 mM at 160 °C
for 10 min using 10% (w/v) slurry content], temperature
[140–200 °C for 10 min using 10% (w/v) slurry content],
hydrolysis time [5–30 min using 10% (w/v) slurry content]
and slurry content [6–16% (w/v)]. HT acid hydrolysis effi-
ciency was calculated using the following equation:
EP (%) = ΔSP ∕TCF × 100, (1)
where EP is pretreatment efficiency using HT acid hydroly-
sis (%), ∆SP the increase in monosaccharide after HT acid
hydrolysis (g/L) and TCF is total carbohydrate and fiber
(g/L).
Enzymatic saccharification was conducted using HT
acid hydrolysate obtained under optimal conditions. The
acid hydrolysate pH was adjusted to 5 using 10 N NaOH.
Various enzymes (16 units/mL), including Cellic CTec2
(120 filter paper units/mL; Novozymes), Viscozyme L (121
β-glucanase U/mL; Novozymes), and their mixture were
added to the 100 mL working volume in a 250-mL flask.
Three enzymes with highly efficient saccharification were
selected for mixed enzyme experiments to identify the opti-
mal conditions for enzymatic saccharification, i.e., 50 °C
on a shaking incubator at 150  rpm. HT acid hydrolysis
13
Bioprocess and Biosystems Engineering
efficiency and enzymatic saccharification were calculated
using the following equation:
EPS (%) = ΔSPS ∕TCF × 100, (2)
where EPS is the efficiency of pretreatment using HT acid
hydrolysis and enzymatic saccharification (%), ∆SPS the
increase in monosaccharide (g/L) during pretreatment using
HT acid hydrolysis and enzymatic saccharification, and TCF
is total carbohydrate and fiber (g/L).
Yeast strain preparation and adaptation to xylose
Saccharomyces cerevisiae KCTC 1126 and P. stipitis KCTC
7228 were obtained from the Korean Collection for Type
Culture (KCTC, Daejeon, Korea). C. lusitaniae ATCC
42720 was obtained from the American Type Culture Col-
lection (ATCC, Manassas, VA, USA). These yeasts were
grown in yeast extract peptone dextrose (YPD) medium
containing 10 g/L yeast extract, 20 g/L peptone and 20 g/L
glucose as a seed culture. The culture was then incubated
with agitation at 150 rpm for 24 h at 30 °C. Each cultured
yeast strain was sampled to determine its dry cell weight
(DCW) by optical density ­(OD600) using the standard curve.
Fermentation was conducted in 250-mL flasks with a work-
ing volume of 100 mL.
Xylose adaptation was then conducted using the three
yeasts. The yeasts were cultured on yeast extract, peptone
and xylose (YPX) liquid medium containing 10 g/L yeast
extract, 20 g/L peptone, and 20 g/L xylose, followed by the
inoculation of 5 mL yeast to 50 mL yeast extract, peptone
and high-concentration xylose (YPHX) medium (10 g/L
yeast extract, 20 g/L peptone, and 60 g/L xylose) for xylose
adaptation. Cells were cultured in ten subcultures on YPHX
during adaptive evolution, and yeast strains were incubated
with agitation at 150 rpm for 24 h at 30 °C. Adapted cells
were washed with fresh medium and transferred to a 250-mL
Erlenmeyer flask containing 100 mL water hyacinth hydro-
lysate for ethanol production.
Ethanol fermentation
Ethanol fermentation was conducted with wild-type or
adapted S. cerevisiae, P. stipitis, or C. lusitaniae with water
hyacinth hydrolysate. Fermentation with the selected yeast
was conducted at 30 °C and 150 rpm for 72 h. Samples were
taken periodically and stored at − 20 °C prior to measure-
ment of ethanol and residual monosaccharides. The ethanol
yield coefficient was calculated using the following equation:
YEtOH = [EtOH]max ∕[Monosaccharide]ini , (3)
where YEtOH is ethanol yield (g/g), [­ EtOH]max the maximum
ethanol concentration achieved during fermentation (g/L),
Bioprocess and Biosystems Engineering
and ­[Monosaccharide]ini is total initial fermentable sugar
(glucose, xylose) concentration (g/L).
Analytical methods
Cell growth was determined based on the ­O D 600 using
an ultraviolet–visible spectrophotometer (Amersham
Biosciences Ultrospec 6300 Pro; Biochrom, Cambridge,
UK). ­O D 600 was converted to dry cell weight (DCW)
using a standard curve of DCW and O ­ D 600 . pH was
measured using a pH meter (CH-8603; Mettler-Toledo
AG, Schwerzenbach, Switzerland). Concentrations of
glucose, xylose, 5-HMF, formic acid, levulinic acid, and
ethanol were determined using high-performance liquid
chromatography (HPLC) (1100 series; Agilent Technol-
ogies, Santa Clara, CA, USA) equipped with a refrac-
tive index detector (RID). An Aminix HPX-87H column
(300 mm × 7.8 mm; Bio-Rad, Hercules, CA, USA) was
used with filtered and degassed 5 mmol/L ­H 2SO 4 as an
eluent at a flow rate of 0.6 mL/min and temperature of
65 °C.
Statistical analysis
Differences in monosaccharide concentration were evalu-
ated by one-way analysis of variance (ANOVA) and Dun-
can’s multiple range test (P < 0.05) using SPSS software
(ver. 23.0; SPSS Inc., Chicago, IL, USA).
Fig. 1  Determination of optimal
conditions for hyper-thermal
(HT) acid hydrolysis in the
degradation of inhibitory com-
pound and monosaccharides.
a ­H2SO4 concentration, b
temperature, c treatment time,
and d slurry content
Results and discussion
Optimization of HT acid hydrolysis
The conditions used to determine the optimal conditions for
HT acid hydrolysis are shown in Fig. 1. Acid concentra-
tion is an important parameter to enhance monosaccharide
production [5]. The results of HT acid hydrolysis at 10%
(w/v) slurry and 160 °C for 10 min, under changing ­H2SO4
concentrations, are shown in Fig. 1a. The highest monosac-
charide production and EP values were obtained at 200 mM
­H2SO4. In comparison, Redding et al. [12] reported that
concentrated acid resulted in the release of high amounts of
monosaccharide. However, levels of inhibitory compounds,
such as 5-HMF, formic acid, and levulinic acid, increased
slightly at acid doses exceeding 200 mM ­H2SO4. Therefore,
200 mM ­H2SO4 was selected as the suitable acid concentra-
tion for HT acid hydrolysis in this study, with an EP value
of 30.7%.
The effects of temperature were evaluated at 10% (w/v)
slurry and 200 mM ­H2SO4 for 10 min as shown in Fig. 1b.
An increase in temperature from 140 to 160 °C yielded an
increase in glucose production. The highest monosaccha-
ride concentrations, including 16.3 g/L glucose and 4.5 g/L
xylose, were obtained at an EP value of 30.7%. However,
upon further increase of temperature to 200 °C, the mono-
saccharide concentration and EP value decreased to 10.3 g/L
and 19.5%, respectively, likely due to the conversion of
monosaccharides to other chemicals such as 5-HMF, and
subsequently HMF into formic acid and levulinic acid [13].
13

At high temperatures (> 160 °C), there was a significant
correlation between monosaccharide degradation and the
formation of inhibitory compounds. Therefore, 160 °C was
chosen as the optimal temperature for this study.
Figure 1c shows the effect of treatment time on monosac-
charide production from water hyacinth biomass. When HT
acid hydrolysis was conducted at 160 °C, with 10% (w/v)
slurry and 200  mM H ­ 2SO4, monosaccharide production
increased as treatment duration increased to 20 min; however,
further increases in treatment duration did not lead to a signifi-
cant increase in monosaccharide production. The optimal HT
acid hydrolysis treatment duration for this study (20 min) was
three times shorter than reported in a previous study [14], and
produced 24.5 g/L monosaccharide with an EP value of 36.2%.
Monosaccharide and HMF concentrations increased as
slurry content increased from 6 to 16% at 200 mM H ­ 2SO4
and 160 °C for 20 min as shown in Fig. 1d. The maximum
monosaccharide concentration at 16% (w/v) slurry content
was 31.1 g/L with an EP value of 32%. However, increasing
the slurry content during HT acid hydrolysis resulted in a
decrease of EP, from 45 to 32%. Therefore, 8% (w/v) slurry
content with an EP value of 45%, was selected for ethanol
production with 0.16 g/L HMF concentration. HMF is the
fermentation inhibitors. However, previous study reported
that HMF inhibition was not active with HMF concentration
lower higher than 1 g/L [15].
Thus, the optimal HT acid hydrolysis conditions were 8%
(w/v) slurry content, 200 mM ­H2SO4, 160 °C and 20 min,
with an EP value of 45%.
Effects of enzymatic saccharification using various
enzymes
Enzymatic saccharification was conducted with various
enzymes to increase monosaccharide concentration before fer-
mentation. The effects of individual enzymes (Cellic CTec 2,
Viscozyme L and Cellic CTec2) and their mixture are shown
in Fig. 2a. A maximum monosaccharide content of 41.7 g/L
was obtained when an enzyme mixture of Cellic CTec 2 and
Viscozyme L was added to the pretreated hydrolysate for
48 h. Ahn et al. [16] reported that mixed enzyme treatment
Fig. 2  Effects of a individual
and mixed enzyme treatments,
and b various activities on
monosaccharide production of
8% (w/v) water hyacinth (Eich-
hornia crassipes) hydrolysate
following HT acid hydrolysis
at pH 5.0 and 50 °C for 48 h.
*The initial glucose and xylose
concentrations were 16.9 and
5.3 g/L, respectively, after HT
acid hydrolysis
13
Bioprocess and Biosystems Engineering
increased the release of monosaccharide compared with sin-
gle enzyme treatment. The effect of an enzyme mixture of
Viscozyme L and Cellic CTec2 was evaluated at 8–24 units/
mL to optimize the enzyme concentration as shown in Fig. 2b.
Monosaccharide concentration did not increase significantly
following the addition over 20  units/mL enzyme. Thus,
20 unit/mL enzyme mixture of Cellic CTec 2 and Viscozyme
L was used as the optimal enzyme concentration for the enzy-
matic saccharification after hyper-thermal acid hydrolysis of
water hyacinth slurry. A monosaccharide concentration and
EPS value were 48.2 g/L and 54.2%, respectively, including
38.0 g/L glucose and 10.2 g/L xylose. Thus, fermentation was
conducted using 48.2 g/L monosaccharide.
Ethanol fermentation with S. cerevisiae, P. stipitis,
and C. lusitaniae
Yeasts in fermentation should be capable of utilizing all
monosaccharides present while withstanding potential inhib-
itors in the hydrolysates. Thus, fermentation was conducted
using S. cerevisiae KCTC 1126, P. stipitis KCTC 7228 and
C. lusitaniae ATCC 42720 to optimize the fermentation con-
ditions as shown in Fig. 3.
Fermentation with S. cerevisiae showed complete utili-
zation of glucose within 48 h, and no utilization of xylose
as shown in Fig. 3a. A previous study reported that S. cer-
evisiae was unable to utilize pentose sugars, which may
comprise up to 40% of water hyacinth hydrolysis [5]. A
maximum ethanol concentration of 15.3 g/L was produced
at YEtOH of 0.32.
P. stipitis can utilize xylose to produce ethanol [17]. The
results of fermentation with P. stipitis in the current study
are shown in Fig. 3b. After fermentation started, glucose
was consumed within 24 h. However, xylose was consumed
slowly until 72 h, leaving 6.8 g/L xylose of the original
10.2 g/L. Passoth et al. [18] also reported that P. stipitis
could ferment xylose to ethanol. However, this yeast prefers
glucose to xylose as a carbon source. The ethanol concentra-
tion and YEtOH after 72 h of fermentation with P. stipitis were
19.5 g/L and 0.41, respectively.
Bioprocess and Biosystems Engineering

Ethanol fermentation with adapted S. cerevisiae,

P. stipites, and C. lusitaniae to high‑concentration
xylose

Adaptive evolution of S. cerevisiae, P. stipitis and C. lusi-

taniae was conducted to enhance ethanol production. Fig-
ure 4 shows fermentation by yeast adapted to high xylose
concentrations.

Fig. 3  Ethanol production by a Saccharomyces cerevisiae, b Pichia

stipitis, and c Candida lusitaniae with 8% (w/v) water hyacinth (E.
crassipes) hydrolysates at 30 °C, 150 rpm for 72 h

Ethanol production using C. lusitaniae resulted in the

complete consumption of glucose within 36 h as shown in
Fig. 3c. Xylose was utilized gradually, with 2.2 g/L xylose
remaining after 72 h of fermentation. C. lusitaniae produced
22.7 g/L ethanol with a Y
­ EtOH of 0.47. C. lusitaniae produce
more ethanol (3.2 g/L) than P. stipitis. A previous study also
reported that C. lusitaniae could ferment xylose better than
Pichia spp [19]. However, none of the three yeasts examined
in the current study could utilize all of the xylose provided. Fig. 4  Ethanol production by a S. cerevisiae, b P. stipitis, and c C.
Thus, adaptive evolution of yeast to high-concentration lusitaniae adapted to high-concentration xylose with 8% (w/v) water
xylose was conducted. hyacinth (E. crassipes) hydrolysate at 30 °C and 150 rpm for 72 h

13
 Bioprocess and Biosystems Engineering

Table 1  Fermentation using Strains Remaining Ethanol (g/L) YEtOH (g/g) Fermenta- Productiv- Y (g/g dry
non-adapted or adapted yeasts sugar (g/L) tion time ity (g/L/h) biomass)
from water hyacinth hydrolysate (h)

S. cerevisiae 10.20 15.30 0.32 60 0.26 0.19

P. stipitis 6.79 1950 0.41 72 0.32 0.24
C. lusitaniae 2.20 22.70 0.47 72 0.32 0.28
Adapted S. cerevisiae 10.20 15.30 0.32 60 0.26 0.19
Adapted P. stipitis 3.70 21.40 0.44 60 0.36 0.27
Adapted C. lusitaniae 0 23.90 0.50 48 0.50 0.30

Fermentation time was set at maximum ethanol production

YEtOH (g/g) = EtOHMAX (g/L)/monosaccharideini (g/L); ethanol yield coefficient, Max ­YEtOH (g/g) = 0.51
productivity (g/L/h) = EtOHMAX (g/L)/fermentation time (h)
Y (g/g) = EtOHMAX (g/L)/water hyacinth content (g/L); ethanol yield per biomass

As shown in Fig. 4a, water hyacinth hydrolysate was fer- concentration of xylose consume every xylose in the water
mented by adapted S. cerevisiae. Glucose was completely hyacinth hydrolysate and fermentation was finished at 48 h
utilized during a 48-h period and converted into ethanol. as shown in Fig. 4c. Therefore, the maximum ethanol pro-
However, S. cerevisiae could not ferment xylose into etha- ductivity (g/L/h) was obtained from adapted C. lusitaniae
nol. Therefore, 10.2 g/L xylose remained and 15.3 g/L etha- with 0.50 g/L/h, which is 1.6-fold higher than wild-type C.
nol was produced with a YEtOH of 0.32. This yield was the lusitaniae (0.32 g/L/h) (Table 1). Thus, adaptive evolution
same as that of the S. cerevisiae wild type. From this result, decreased the fermentation time and led to high ethanol
we determined that xylose could not be utilized by wild S. productivity. A previous study also reported that adapted
cerevisiae as well as by S. cerevisiae adapted to xylose. Sev- C. lusitaniae enhanced the monosaccharide uptake rate
eral other studies have produced ethanol from xylose using and produced a higher ethanol concentration than the wild-
genetically engineered S. cerevisiae [20–22]. type C. lusitaniae [14]. Previous studies have examined
Pichia stipitis adapted to high-concentration xylose was ethanol production yields from water hyacinth hydrolysate
then used to ferment water hyacinth hydrolysate Fig. 4b.
Glucose was completely consumed after 36 h and xylose
was slowly utilized until the end of the fermentation period.
Agbogbo et al. [23] reported that repression of xylose uptake
occurs in fermentation media containing glucose and xylose,
due to the low-affinity transport system shared between
glucose and xylose. Glucose inhibits xylose transport by
noncompetitive inhibition [24]; therefore, enhancement of
xylose consumption and high yield of ethanol were obtained
from P. stipitis after adaptive evolution, with a maximum
ethanol concentration of 21.4 g/L and YEtOH of 0.44. The
xylose consumption rate and yield of adapted P. stipitis were
2.0 and 1.1 times higher than those of the wild-type P. stipi-
tis. However, 3.7 g/L xylose remained following fermenta-
tion. Thus, C. lusitaniae was used to consume the remainder
of the xylose.
Water hyacinth was then fermented using adapted C.
lusitaniae from high-concentration xylose as shown in
Fig. 4c. Glucose and xylose were completely utilized after
36 and 48 h, respectively, producing a maximum ethanol
concentration of 23.9 g/L and ­YEtOH of 0.50 after 48 h.
Adapted C. lusitaniae produced more ethanol than wild-
type C. lusitaniae due to the consumption of all monosac-
charides in the broth. Wild type of C. lusitaniae originally
converts xylose to ethanol better than wild-type S. cerevi- Fig. 5  Mass balance flow chart of bioethanol production from water
siae and P. stipitis. However, adapted C. lusitaniae to high hyacinth

13
Bioprocess and Biosystems Engineering
using different approaches [25, 26]. Palmqvist et al. [27]
reported that yeast adaptation could be ascribed to the syn-
thesis of enzymes or cofactors for efficient sugar metabo-
lism and reduction of damage from inhibitors. Therefore,
enhanced ethanol production in high-concentration xylose
media was achieved by improved xylose utilization.
The overall mass balance is described in Fig. 5. The
maximum ethanol concentration was 23.9 g/L with YEtOH
coefficient of 0.32 from 8%(w/v) of water hyacinth after
HT acid hydrolysis and enzymatic saccharification. These
results indicated that the ethanol yield from water hyacinth
achieved 0.30 g ethanol/g of dry material, which increased
1.57–6.80-fold higher than that from water hyacinth on
previous studies [3, 28]. Kang et al. produced 69.2 g/L
ethanol from 25% (w/v) of lignocellulosic biomass, Mis-
canthus sacchariflorus [29]. However, the yield from M.
sacchariflorus was 0.27 g ethanol/g of dry material. There-
fore, water hyacinth can obtain high ethanol concentra-
tion with increasing the biomass content. These optimal
fermentation profiles provide a basis for maximal ethanol
production from the water hyacinth via optimal HT acid
hydrolysis, enzymatic saccharification and yeasts adapted
to high-concentration xylose.
Conclusions
Water hyacinth is used as biomass for ethanol production
due to its high cellulose content and low amounts of lignin.
HT acid hydrolysis pretreatment and enzymatic saccharifi-
cation were conducted under optimal conditions. A maxi-
mum of 48.2 g/L monosaccharide was obtained from 8%
(w/v) slurry content. HT acid hydrolysis and enzymatic
saccharification enhanced monosaccharide production.
Fermentation was conducted with wild-type and adapted
S. cerevisiae, P. stipites, and C. lusitaniae. Neither wild
nor adapted S. cerevisiae could not utilize xylose. Fermen-
tation with adapted P. stipitis and C. lusitaniae produced
higher ethanol concentrations from xylose than the wild
type due to adaptive evolution, which reduced glucose
repression in xylose consumption. An ethanol concentra-
tion of 23.9 g/L and Y EtOH of 0.50 was achieved using
adapted C. lusitaniae. The monosaccharide conversion
efficiency achieved through pretreatment and ethanol yield
from fermentation was higher in the present study than
reported for many types of lignocellulosic feed stocks.
Acknowledgements  This research was supported by Basic Sci-
ence Research Program through the National Research Foun-
dation of Korea (NRF) funded by the Ministry of Education
(2016R1D1A1A09918683).
Compliance with ethical standards 
Conflict of interest  The authors declare that they have no conflict of
interest.
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