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Effect of Various Pretreatment Methods on Sugar andEthanol Production from Cellulosic Water Hyacinth
Shahabaldin Rezania,a* Hossein Alizadeh,b Junboum Park,a* Mohd Fadhil Md Din,cNegisa Darajeh,d Shirin Shafiei Ebrahimi,e Bidyut Baran Saha f,g and Hesam Kamyab hEffects of acid, alkali, ionic liquid (IL), and microwave-alkali pretreatmentson cellulosic water hyacinth (WH) were investigated based on the total
reducing sugars (TRS) and ethanol production. For the first time, IL
pretreatment with (1-Ethyl-3-methylimidazolium acetate ([EMIM][Ac]) wasused for WH, and the efficiency was compared with the other methods.
Cellulase and Saccharomyces cerevisiae were fermented together for 72
h. Based on the results, all pretreatment methods effectively increased thesugar content as well as the ethanol yield. Untreated WH had 25 ± 1.5
mg/g of TRS, which was increased to 157 ± 8.2 mg/g, 95 ± 3.1 mg/g, 51 ±4.2 mg/g, and 45 ± 2.6 mg/g via alkali, microwave-alkali, acid, and IL
pretreatments, respectively. The highest TRS level of 402 mg/g was
obtained in 24 h and 6.2 ± 0.4 g/L of ethanol in 48 h of fermentation withthe alkali-treated WH. The ethanol production was followed by othertreatment methods of WH in the order of microwave-alkali, acid, and IL.
The results indicated that the ethanol production from WH was related tothe type of pretreatment as well as the TRS production.
Keywords: Water hyacinth; Ethanol production; Total reducing sugar; Pretreatment methods
Contact information: a: Department of Civil and Environmental Engineering, Seoul National University,
Seoul, Republic of Korea; b: Bio-Protection Research Centre, Lincoln University, Lincoln 7647, New
Zealand; c: Centre for Environmental Sustainability and Water Security (IPASA), Research Institute forEnvironmental Sustainability, Universiti Teknologi Malaysia, 81310, Johor Bahru, Malaysia; d: School of
Biological Sciences, University of Canterbury, Christchurch 8140, New Zealand; e: School of Education,
Universiti Teknologi Malaysia, Johor Bahru, Malaysia; f: International Institute for Carbon-Neutral
Energy Research (WPI-I2CNER), Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan;
g: Mechanical Engineering Department, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395,
Japan; h: Engineering department, Razak faculty of technology and Informatics, Universiti Teknologi
Malaysia, Jalan sultan Yahya Petra, 56100, Kuala Lumpur;* Corresponding authors: shahab_rezania89@yahoo.com; junbpark@snu.ac.kr

INTRODUCTION
Fossil fuel depletion in the near future can be predicted by the continuous overutilization of natural energy sources (Jambo et al. 2016). Agricultural products, such as
corn, wheat, and sugarcane, are defined as the first generation of raw materials forbioethanol production. Likewise, lignocellulosic biomass is the second generation, andalgal biomass is the third generation (Baeyens et al. 2015). This energy source also has thepotential to reduce greenhouse gas emissions originating from fossil fuels and by carbon
sequestration (Tye et al. 2016). The use of lignocellulosic biomass as the feedstock forfermentable sugar production is the key element in sustaining the production of bioproducts
like bioethanol (Mandade et al. 2016).

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Water hyacinth is a type of lignocellulosic material that is potentially available intropical regions (Sing and Bishnoi 2013; Rezania et al. 2015). In an untreated biomass,
cellulose, hemicelluloses, and lignin are linked to form a strong structure that is difficult toprocess (Rezania et al. 2017). Because the lignin content of the lignocellulose can affectthe ethanol production process, the selection of suitable biomass is important. The WH has
high carbohydrate and low lignin content, an impressive growth rate, and no competitionon land, which makes it a suitable lignocellulosic material for bioenergy generation(Rezania et al. 2016). The economic potential of biofuel production can be improved byusing low-lignin biomass in comparison to rich lignin biomass (Bhatt and Shilpa 2014).
According to Singh and Bishnoi (2012), there are three main operations needed to convert
lignocellulose to bioethanol: pretreatment (necessary to liberate cellulose andhemicellulose), cellulose hydrolysis to produce fermentable sugars, and the fermentationof reducing sugars. In this way, the most common and traditional microorganism used inindustrial bioethanol production is the yeast Saccharomyces cerevisiae (Dionisi et al.
2015).
A major challenge for bioethanol production is finding a cost-effective pretreatmentof lignocellulosic biomass (Singh et al. 2015). Different cost-effective pretreatment
methods have been identified based on different types of lignocellulosic biomass andproductivity (Srivastava et al. 2015). The effectiveness of these pretreatments depends on
the physical structure and chemical composition of the biomass, as well as the pretreatment
conditions (Sarto et al. 2019). The choosing of a pretreatment method can determine thesuccess in overcoming this complicated structure and the factors that constrain the
enzymatic hydrolysis of the lignocellulosic biomass (Sun et al. 2016). For instance, tocatalyze the polysaccharide in the lignocelluloses, cellulase can serve an important
catalytic enzyme with appropriate levels of stability in many ILs (Elgharbawy et al. 2016).
In addition, thermal pretreatment, mild alkali pretreatment, and microbial combined diluteacid pretreatment are new approaches for ethanol production from cellulosic WH (Barua
and Kalamdhad 2017; Narra et al. 2017; Zhang et al. 2018).
To increase the bioethanol yield, various pretreatment methods are applied todecrease the recalcitrance of lignocellulosic feedstock (Zheng et al. 2014). The supernatantfrom the lignocelluloses enzymatic hydrolysis contains both hexoses and pentoses sugars(Rezania et al. 2018). As demonstrated by Cheng et al. (2014), the simultaneous processesof pretreatment and wet storage conserve 70% of carbohydrates and remove 40% of lignin
with 325 mg/g reduced sugar yield from the WH biomass. Therefore, the aim of this study
is to evaluate the effectiveness of acid, alkali, IL, and microwave-alkali pretreatments on
the ethanol and total reducing sugars production from WH based on fermentation time.
Meanwhile, the main novelty of this study was to use IL pretreatment with 1-ethyl-3methylimidazolium acetate ([EMIM][Ac]) for WH which the efficiency was comparedwith the mentioned methods.

EXPERIMENTAL
Materials
Water hyacinth preparation
The plants were collected from a tank designed for propagation in theenvironmental lab (IPASA) at the Universiti Teknologi Malaysia, Johor. The WH leaves
were separated and washed with distilled water, chopped, oven-dried, and then stored in
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plastic bags for further usage. The chemicals used in this study, such as NaOH and H2SO4,
were analytical grade reagents. The ionic liquid solution was bought from Sigma AldrichCompany (Kuala Lumpur, Malaysia).
Methods
Different pretreatment methods
In this study, four types of pretreatment methods were performed using acid, alkali,
IL, and microwave-alkali. For the alkali treatment, 10 g of dried WH were soaked in an
alkali solution for 1 h followed by a treatment time of 10 min at 150 °C inside of a furnace.
Then, the hydrolysate was collected via a filtration method (Ahn et al. 2012). For the acidtreatment, 10 g of WH were mixed with 90 mL of 4% dilute sulfuric acid (W/V) and thenautoclaved at 120 °C for 15 min. Then, the treated biomass was neutralized with an additionof calcium hydroxide (3N) and filtered using a membrane filter (0.45 µm). The solid
residues after pretreatment were washed with deionized water, then dried and stored for
enzymatic saccharification (Fileto-Pérez et al. 2013).
The IL pretreatment was performed by mixing 51.0 mL of 1-ethyl-3methylimidazolium acetate ([EMIM][Ac])) with 3 g of WH in a 17:1 ratio. The solution
was heated and stirred at 120 °C and incubated for 180 min. To regenerate the WH, equalvolumes of deionized water were added to the WH/solvent solution, which made theprecipitation occur quickly. The sample was centrifuged and the supernatant containing IL
was removed. Next, the precipitate was washed using deionized water. Finally, theregenerated WH was filtered using Whatman filter paper and oven-dried at 60 °C for 48 h
in a vacuum (Gao et al. 2013a). The microwave-alkali pretreatment was conducted in a
WX-4000 microwave digestion system (Hangzhou, China). In this pretreatment, the WH
was treated with 1.0% (w/v) NaOH solution for 24 h at 45 °C in a shaker at 120 rpm, thenheated for 1.0 min at 420 w in a microwave oven (SANYO Super Shower Wave 900W,
Sanyo Denki America, Inc., Torrance, CA, USA) (Xia et al. 2013). FTIR analysis was
performed using (Nicolet iS5, Thermo Fisher Scientific, Inc., USA). The spectra wereobtained in the transmission mode and the range of 600 to 4000 cm −1 at a resolution of 4
cm−1 with 32 scans per each sample.
Preparation of yeast as inoculum
A total of 5 g of yeast, Saccharomyces cerevisiae (S. cerevisiae) was added to 50
mL of distilled water and incubated at 37 °C with 150 rpm for 24 h. The potato dextrose
agar medium (PDA) was prepared to culture the yeast cells. Then, S. cerevisiae wasstreaked on the PDA plates to grow the colonies and was kept in an incubator at 37 °C for
24 h. To enhance the growth rate and activity of S. cerevisiae, the best colonies in terms ofsize were screened and transferred to 50 mL of nutrient broth (NB) in a 250-mL Erlenmeyer
flask at 30 °C.
Later, the plates were transferred to the shaker incubator in 37 °C for three days. A
loopful of the colony was transferred to the test tube and mixed in 50 mL of distilled water.
The tubes were centrifuged in 4000 rpm for 20 min (the temperature was held at -4 °C).
The top layer (supernatant) was discarded and the pellet was collected to measure theoptical density (OD) at 600 nm using a spectrophotometer (HACH DR6000; Loveland,
CO, USA). The S. cerevisiae suspension cultures were used as an inoculum forfermentation (Mukhopadhyay and Chatterjee 2010).

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Simultaneous sacchariﬁcation and fermentation (SSF)
The SSF method was used for the enzymatic hydrolysis and fermentation of thetreated WH biomass to produce ethanol. This process consisted of 5 g of the treated WH(previously autoclaved for 15 min at 121 °C), ﬁlter-sterilized cellulase (extracted from
Aspergillus niger) solubilized in 0.1 M sodium phosphate, and 10 mL of S. cerevisiae in a
Mendel medium. The initial pH of the fermentation medium was adjusted to 5.0, and the
SSF was carried out at 37 °C in 250-mL conical ﬂasks with 80 mL of fermentation medium
and a final volume of 100 mL with agitation for 72 h. The sampling was completed at 12
h and approximately 10 mL of each sample was taken. The samples were centrifuged at4000 rpm for 15 min and the pallet was discarded. The supernatant was separated and used
to determine the ethanol and reducing sugar content (Eshtiaghi et al. 2012).
Analytical procedures
The WH composition (cellulose, hemicellulose, and lignin content) was measured
according to Di Blasi et al. (1999). The 3,5-dinitrosalicylic acid (DNS) method was used
to determine the total sugar reduction in the enzymatic hydrolysate of the acid-treatedbiomass (Miller 1959). The amount of ethanol during fermentation was measured using
gas chromatography (GC) (Model 7890, Hewlet Packard, Waldbronn, Germany) with an
FID detector and column HP20 M. The temperatures of the oven, column inlet, and detector
were 200 °C, 300 °C, and 300 °C, respectively. The carrier gas was helium with a flow rateof 25 mL/min (Ang and Abd-Aziz 2015). The ethanol concentration in the samples was
identified by comparing the area of the respective sample against the area of a reference
sample containing only the standard ethanol. All of the experiments were conducted intriplicate. The theoretical yield value and volumetric ethanol productivity were calculated
based on Eqs. 1 to 3:
Volumetric ethanol productivity (Qp) =Theoretical Yield Value (ή, %) =g

Ethanol Yield (g) =Ethanol Concentration
Fermentation Time

Ethanol Yield
× 100
0.51

Fermentation Medium (mL)× Ethanol Concentration
Gram Biomass ×1000

(1)
(2)

(3)

Theoretical maximum reducing sugar and ethanol yield
For the calculation of the theoretical maximum reducing sugar yield, it was
assumed that the cellulose and hemicellulose in the WH were completely hydrolyzed into
hexose (glucose) and pentose (xylose). The weight ratio of the glucose molecule tocellulose was 1.111 (180/162), whereas that of xylose to hemicellulose was 1.136(150/132). The reaction formula of cellulose conversion transformed into glucose, and thehydrolyze percentage was calculated based on Hu and Wen (2008).

RESULTS AND DISCUSSION
Composition of Untreated WH
The untreated WH consisted of 16.4 ± 0.35% cellulose, 32.7 ± 0.14%
hemicellulose, 5.7 ± 0.05% lignin, and 10.7 ± 0.1% ash content. This demonstrated that
WH could be a favorable carbohydrates source. However, Table 1 shows that previous
studies obtained different amounts of carbohydrates and lignin from the WH biomass.
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Various geographic conditions, different parts of the WH, different growth states, andmethods for drying biomass could be reasons behind these differences.
Table 1. Chemical Composition of Untreated Water Hyacinth
Compositionof WH(Gao etal. 2013b)(Cheng etal. 2014)(Zhang etal. 2015)(Das etal. 2016)(Ruan et al.
2016)

This Study

Cellulose (%)

18.3

24.15

18.07

24.7

24.5

16.4 ± 0.35

Hemicellulose
(%)

23.3

27.23

28.21

32.2

34.1

32.7 ± 0.14

Lignin (%)

17.7

12.39

7.03

3.2

8.6

5.7 ± 0.05

Total reducing sugar from different pretreatment methods
Manivannan and Narendhirakannan (2015) observed that the amount of glucose,
xylose, and TRS range from 7 mg/g to 41 mg/g of WH. To evaluate the effectiveness ofdifferent pretreatment methods, the TRS was measured before and after pretreatment, asshown in Table 2.
Table 2. Reducing Sugars by Different Pretreatment Methods
WH Biomass

Untreated

Acid TreatedAlkali Treated

IL TreatedMicrowavealkali TreatedTotal Reducing
Sugars (mg/g ofWH)

25 ± 1.5

51 ± 4.2

157 ± 8.2

45 ± 2.6

95 ± 3.1

The amount of total reducing sugars (hexoses and pentoses) released from thesubstrate equated to the effectiveness of the pretreatment method (Das et al. 2016). Basedon Table 2, the highest rate of TRS obtained was from the alkali pretreatment at 157 mg/gof WH. This pretreatment enhanced the sugar ratio more than six times more in comparison
to the untreated WH (25 mg/g). Similarly, Harun et al. (2011) obtained 24.7 mg sugar/g
dry matter of sugars in untreated WH. In a study by Xu et al. (2016), 99 mg/g yield of TRS
from untreated WH was produced during hydrolysis. The acid, IL, and microwave-alkali
pretreatments produced 51 mg/g, 45 mg/g, and 95 mg/g WH of TRS, respectively. In
conclusion, the highest TRS was obtained by the alkali-treated WH, followed by
microwave-alkali-, acid-, and IL-treated WH, respectively.
TRS production during fermentation
During fermentation, the majority of reducing sugars were converted to ethanol
through the S. cerevisiae activity. Initially, the TRS was increased by the cellulase activity,
and then decreased due to the sugar consumption by S. cerevisiae to produce ethanol. The
high TRS production was due to breakage of the cellulosic structure by the cellulase, which
resulted in the exposure of more sugars to microorganisms. In addition, the treated biomassin all pretreatment methods showed an increase in sugar amount in comparison to theuntreated biomass. The pretreatments were effective in increasing the TRS during
fermentation for ethanol production.
TRS from alkali and microwave-alkali treated WH
Based on Fig. 1a and Table 2, the highest reducing sugar was obtained from thealkali-treated WH. Initially, 157 mg/g of reducing sugars were obtained from the alkaliRezania et al. (2019). “Pretreatment of water hyacinth,” BioResources 14(1), 592-606.

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treated sample and then were increased up to 402 mg/g after 24 h of fermentation. The
reduction was observed afterwards due to the sugar consumption by the microorganisms
until the end of the experiment. Similarly, Ganguly et al. (2013) observed 465 mg/g of TRS
after an alkali pretreatment of WH. The high level of TRS was due to the availability oflarge amounts of monosaccharide (cellulose and hemicellulose) after the alkali
pretreatment.

a

b

450

400
350

350

Reducing Sugars (mg/g)

Reducing Sugars (mg/g)

400

300
250
200
150
100
50

300
250
200
150
100
50
0

0
0

12

24

36

48

Fermentation (h)

60

72

0

12

24

36

48

60

72

Fermentation (h)Fig. 1. TRS of alkali-treated and microwave-alkali-treated WH in fermentation: a) alkali-treated
and b) microwave-alkali-treated

As shown in Fig.1b, a sharp increment of TRS occurred from 95 mg/g to 346 mg/g
in 36 h by the microwave-alkali-treated WH. The radiation successfully enhanced theamount of reducing sugars in the WH biomass. The TRS decreased sharply until the endof the fermentation at 72 h based on the S. cerevisiae activity in the fermentation flask andreached 144 mg/g at 72 h. Meanwhile, Zhang et al. (2015) obtained only up to 99.12 mg/g
of TRS from the microwave-alkali-treated WH. Aswathy et al. (2010) observed the
maximum reducing sugar yield from the hydrolysis of alkali-treated WH, which was 639.4
mg/g. In a study by Xia et al. (2013), the maximum reducing sugar yield of 483 mg/g fromWH substrate, with 94.6% of the theoretical reducing sugar yield, was obtained using a
microwave-alkali pretreatment. As stated by Singh and Bishnoi (2013), the alkali-treated
WH has a higher amount of monosaccharide compared to the acid and oxidative
pretreatment. They observed the reducing sugar amount was 5.1 g/L, 6.2 g/L, 9.3 g/L, 13.5
g/L, 11.3 g/L, and 8.8 g/L after 12 h, 24 h, 48 h, 60 h, 72 h, 96 h, and 120 h of fermentation,
respectively.
TRS from acid and IL treated water hyacinth
As shown in Fig. 2a, the highest TRS from acid-treated WH was obtained after 48
h of fermentation at 236 mg/g of WH. After 48 h, the TRS was reduced to 62 mg/g at 72 h
fermentation, which implied sugar consumption by microbes. Zhang et al. (2015) obtained
197.60 mg/g of TRS in hydrolysates from the acid-treated WH. Meanwhile, 3.79 g/L to
5.27 g/L of total reducing sugars was obtained from the alkali-treated WH using S.
cerevisiae during fermentation (Manivannan and Narendhirakannan 2015).

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b
300

300

250

250

Reducing Sugar (mg/g)

Reducing Sugar (mg/g)

a

200
150
100

200
150
100
50

50

0

0
0

12

24
36
48
Fermentation (h)

60

72

0

12

24
36
48
Fermentation (h)6072Fig. 2. TRS of acid-treated and IL-treated WH in fermentation: a) acid-treated and b) IL-treated

Similar to other pretreatment methods, the reduction of TRS in the IL-treatedsample occurred between 48 h and 72 h. During this stage, the depletion of sugars occurredin the fermentation due to microorganism activity. The highest TRS was 164 mg/g at 48 h,and it was reduced to 68 mg/g at the end of fermentation at 72 h (Fig. 2b). In this study,
only 164 mg/g of TRS were obtained from the IL-treated WH, which could have been
related to the level of lignin in the substrate or type of IL. In contrast, high levels of TRS
at 584 mg/g were obtained from that IL-treated WH 1-butyl-3-methylimidazolium acetate(BMIMA) (Guragain et al. 2011).
FTIR analysis
For better validation of the effects of various pretreatment methods in the structureof WH, the analysis of FTIR spectra before and after the pretreatment is shown in Fig. 3.
After alkali pretreatment of WH, in the range of 1000 cm-1 to 1500 cm-1, five peaks wereappeared 1025 cm-1, 1154 cm-1, 1315 cm-1, 1408 cm-1, and 1415 cm-1 (Fig. 3a). These peaks
represented the structural features of cellulose and hemicelluloses in WH biomass. Basedon the results of this study, the alkali pretreatment increased the amount of cellulose andhemicellulose in WH. In addition, a significant drop of band intensity was observed at
2915-1, which indicated the reduction in the hydrogen bonding of cellulose in WH structure.
In contrast, the absorption band at 2940 cm−1 after alkali pretreatment and enzymatichydrolysis of WH was observed, as reported by Das et al. (2015)
Based on FTIR spectra of acid treated WH, no peaks in the range of > 1000 wereobserved. This means that the acid pretreatment only increased the cellulose of WH and
was not effective in lignin removal (Fig. 3b). Although, Cheng et al. (2014) found the peakintensity of 894 cm-1, which confirms the increase of cellulose after acid treatment of WH
due to the removal of amorphous hemicellulose. In addition, because of the completehydrolysis of hemicellulose, the peak intensity of 1739 cm-1 was remarkably decreased
after acid pretreatment. FTIR spectra showed significant reduction of band intensity at
1321 cm-1 (C–H deformation in hemicelluloses) due to the removal of hemicelluloses.

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a

c

b

d

Fig. 3. FTIR analysis of different pretreatments a) Alkali treated WH b) Acid treated WH c)
Microwave-alkali treated WH d) IL treated WH

FTIR spectra of microwave-alkali treated WH showed that the disruption of lignin
structure (Fig. 3c). Furthermore, the peak intensity of 1630 cm-1 in C=O of acetyl groupsof lignin dropped due to the lignin cleavage. Furthermore, it also confirmed that the
microwave irradiation was effective for complete hydrolysis of tough WH structure (deSouza Moretti et al. 2014).
In FTIR spectra of IL treated WH there was a sharp band at 774 cm−1, whichcorresponds to the C1 group frequency and can be attributed to the β-glycosidic linkages(1→4) between xylose units in hemicelluloses. Another peak appeared at 1,240 cm-1
corresponding to (C–O stretching in lignin and hemicellulose), which was indicative of theremoval of hemicellulose (Fig. 4d).
Similarly, 2922, 1427, and 1373 cm-1 bands were related to the characteristic ofcellulose and did not substantially change due to the protection of cellulose during IL
pretreatment (Xia et al. 2013). Furthermore, IL pretreatment had more contribution in
disruption of β-glycosidic linkages and hemicelluloses reduction. Recently, Singh et al.
(2018) showed that treatment of WH using different imidazolium-based ILs such as [1alkyl-3-methylimidazolium bromide, [Cnmim]Br, resulted in high levels of cellulose
disruption.

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Ethanol Production from Differently Treated WH
Ethanol production from alkali- and microwave-alkali treated WH
Alkali and microwave-alkali pretreatment are two effective methods for ethanol
production from WH. Table 3 shows a summary of some studies that used alkali treated
WH in different NaOH concentration by using S. cerevisiae.
Table 3. Ethanol production from alkali-treated WH in different studies
NaOH
concentration

Yeast type

Fermentation(h)Ethanol yield(g/L)

Reference

1.0%

S. cerevisiae
andPachysolentannophilus

48

5.2

(Mukhopadhyay
and Chatterjee
2010)

5.0%

S. cerevisiae

36

4.3 ± 0.8

5.0%

S. cerevisiae

48

3.07

5.0%

S. cerevisiae

72

6.76

(Singh and Bishnoi
2013)(Ganguly et al.
2013)(Das et al. 2015)

As shown in Fig. 4a and Table 2, the alkali-treated WH had the highest TRS at 402
mg/g and the highest ethanol was produced at 48 h of fermentation by 6.2 ± 0.4 g/L.
ba
Ethanol concentration

400

500

9

450

Total reducing sugars

400

Ethanol concentration

8

350

7

300

6

250

5

200

4

150

3

100

2

50

1

0

0
0

12

24

36

48

Fermentation (h)

60

72

7
6

350

5

300

4

250
200

3

150

2

100

1

50

0

0

0

12

24

36

48

60

72

Fermentation (h)Fig. 4. Ethanol production: a) alkali-treated WH and b) microwave-alkali-treated WH

Das et al. (2015) found that the higher ethanol yield by P. stipitis was due to theutilization of both pentose and hexose. Ethanol production based on sugar consumption in
the alkali-treated WH is shown in Fig. 4a. The ethanol production from the microwavealkali-treated WH based on TRS is shown in Fig. 4b. By using microwave-alkali-treated
WH, 4.3 ± 0.5 g/L of ethanol with an ethanol yield of 0.86 g/5g of WH and 0.059 ofvolumetric ethanol productivity in 48 h of fermentation was produced. Meanwhile, thehighest amount of TRS was 346 mg/g in 36 h. The ethanol production via microwavealkali increased sharply after 24 h to 48 h. Therefore, the constant reduction occurred dueto the low rate of TRS production during fermentation.

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Ethanol Concentration (g/L)

Reducing Sugars (mg/g)

450

10

Reducing Sugars (mg/g)

Total reducing sugars

Ethanol Concentration (g/L)

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Ethanol production from acid and IL treated WH
Figure 5a shows the ethanol production from the acid-treated WH based on TRS
and fermentation time. The acid-treated WH produced 1.8 ± 0.8 g/L of ethanol with a 0.025
productivity and yield of 0.03 g/g of WH in 60 h of fermentation. In addition, the sugar
level was drastically decreased because of the TRS consumption by the microbes forethanol production. In this study, 4% (w/v) H2SO4 was applied and 236 mg/g of TRS wereobtained at 36 h of fermentation. In previous studies, 33.3 g/L of total sugars were obtainedin optimized conditions by Reales-Alfaro et al. (2013) and 21 g/g by Nigam (2002) after
pretreatments with sulfuric acid. These findings further support the idea that the amount ofsugars released from the substrate depends on the pretreatment method efficiency and the
enzyme activity. As cellulase cleaves the cellobiose units to liberate glucose due to high βglucosidases activity about 21.39 (U/gDS), it effectively enhanced the TRS production andsubsequently some end products such as ethanol. As reported by Alvira et al. (2010), thepresence of ILs residues could have a significant negative effect on cellulase activity.
Therefore, to prevent the decrease of final sugars concentrations, ILs removal is necessary.

450
350

Ethanol concentration

6
5

300

4

250
200

3

150

2

100

1

50

0

0

0

12

24

36

48

60

72

Reducing Sugars (mg/g)

400

Total reducing sugars

Ethanol Concentration (g/L)

Reducing Sugars (mg/g)

b

7

500
450
400
350
300
250
200
150
100
50
0

7
Total reducing sugars

6

Ethanol concentration

5
4
3
2
1
0
0

12

Fermentation (h)

24

36

48

60

72

Fermentation (h)Fig. 5. Ethanol production: a) acid-treated WH and b) IL-treated WH

In this study, the lowest ethanol yield and TRS production were obtained from theIL-treated WH. Hence, there are not many published studies using IL pretreatment forethanol production from WH. It could hinder the enzymatic hydrolysis as well, as reported
by Guragain et al. (2011). The reason could be due to a high moisture content of WH,
which leads to a reaction of water molecules with IL solvents during fermentation. Besides,
some small molecules such as fructose and lignin can be dissolved in IL solutions during
the pretreatment process (Gao et al. 2013a).
Based on Fig. 5b, the IL-treated WH produced 1.3 ± 0.9 g/L of ethanol with avolumetric productivity of 0.018 and a yield of 0.02 g/5g of WH. The highest ethanol
production was obtained at 60 h, and the highest TRS was obtained at 48 h. As
demonstrated by Guragain et al. (2011), by using a dilute acid pretreatment on WH, thehydrolysis yields of glucose and total reducing sugars were 2.1 and 3.3 times higher thanIL pretreatments. Glucose and total sugars yield of the IL pretreatments were 332 mg/g and
584 mg/g of WH, respectively. One of the reasons for such results could have been due tothe solubilization of the majority of all hemicelluloses in dilute acid pretreatments.

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Ethanol Concentration (g/L)

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Theoretical maximum reducing sugar and ethanol yieldIn this study, a maximum theoretical yield of reducing sugar for untreated WH was(18.2 + 37.2) = 0.554 g/g of untreated WH biomass. Theoretically, 0.51 kg of ethanol per
kilogram can be obtained from glucose and xylose. Therefore, the maximum theoreticalethanol yield of WH was (0.554 × 0.51) = 0.282 g/g of raw biomass of WH. However, Daset al. (2015) state the maximum theoretical ethanol yield as 0.3659 g/g of WH, which was
0.294 g/g of WH as reported by Cheng et al. (2014). This is comparable to ethanol yields
of other lignocellulosic materials, such as rice straw with 0.326 g/g (Imman et al. 2015).
Table 4 shows the ethanol productivity and yield by different types of pretreatment
methods. Conditions of fermentation, species, and carbohydrate content of WH notably
contributed to ethanol production.
Table 4. Ethanol Production from Differently Treated WH
Different
Pretreatments
AlkaliMicrowave-alkali
Acid
Ionic Liquid

HighestLevel of
TRS (g/g)
402
346
234
164

HighestEthanol (h)
48
48
60
60

Ethanol
Production(gp/L)
6.2 ± 0.4
4.3 ± 0.5
1.8 ± 0.8
1.3 ± 0.9

Volumetric
Ethanol
Productivity
0.086
0.059
0.025
0.018

Yield (gEthanol/ 5
g WH)
0.12
0.086
0.03
0.02

CONCLUSIONS
1. The IL pretreatment with 1-ethyl-3-methylimidazolium acetate ([EMIM][Ac]) was noteffective for sugar and ethanol production from WH.
2. The most effective pretreatment method was alkali followed by microwave-alkali, acid,and IL.
3. FTIR analysis showed a decrease of the band at 1430 cm-1 in all treated samples, which
confirmed the effective disruption of the crystalline structure of WH.
4. The highest ethanol yield was 6.2 ± 0.4 g/L via alkali-treated biomass with 0.086
volumetric ethanol productivity and an ethanol yield of 0.012 g/5g of WH in 48 h.
5. The acid, microwave-alkali, and IL-treated WH produced 4.3 ± 0.5 g/L, 1.8 ± 0.8 g/L,
and 1.3 ± 0.9 g/L of ethanol, respectively.

ACKNOWLEDGMENTS
This research was supported by a grant (2016R1D1A1A02937267) provided by
Korean National Research Foundation (KNRF) which is part of project individual basic
science & engineering research program (SGER).

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REFERENCES CITED
Ahn, D. J., Kim, S. K., and Yun, H. S. (2012). “Optimization of pretreatment andsaccharification for the production of bioethanol from water hyacinth by
Saccharomyces cerevisiae,” Bioprocess Biosystem Engineering 35(1-2), 35-41. DOI:
10.1007/s00449-011-0600-5
Alvira, P., Tomás-Pejó, E., Ballesteros, M. J., and Negro, M. J. (2010). “Pretreatment
technologies for an efficient bioethanol production process based on enzymatichydrolysis: a review, “Bioresource Technology 101(13), 4851-4861. DOI:
10.1016/j.biortech.2009.11.093
Ang, S. K., and Abd-Aziz, S. (2015). “Potential uses of xylanase-rich lignocellulolytic
enzymes cocktail for oil palm trunk (OPT) degradation and lignocellulosic ethanol
production,” Energy & Fuels 29(8), 5103-5116. DOI:
10.1021/acs.energyfuels.5b00891
Aswathy, U. S., Sukumaran, R. K., Devi, G. L., Rajasree, K. P., Singhania, R. R., andPandey, A. (2010). “Bio-ethanol from water hyacinth biomass: An evaluation of
enzymatic saccharification strategy,” Bioresource Technology 101(3). 925-930. DOI:
10.1016/j.biortech.2009.08.019
Baeyens, J., Kang, Q., Appels, L., Dewil, R., Lv, Y., and Tan, T. (2015). “Challenges andopportunities in improving the production of bio-ethanol,” Progress in Energy andCombustion Science 47, 60-88. DOI: 10.1016/j.pecs.2014.10.003
Barua, V. B., and Kalamdhad, A. S. (2017). “Effect of various types of thermal
pretreatment techniques on the hydrolysis, compositional analysis andcharacterization of water hyacinth,” Bioresource Technology 227, 147-154. DOI:
10.1016/j.biortech.2016.12.036
Bhatt, S. M., and Shilpa (2014). “Lignocellulosic feedstock conversion, inhibitor
detoxification and cellulosic hydrolysis: A review,” Biofuels 5(6), 633-649. DOI:
10.1080/17597269.2014.1003702
Cheng, J., Wang, X., Huang, R., Liu, M., Yu, C., and Cen, K. (2014). “Producing ethanolfrom water hyacinth through simultaneous saccharification and fermentation withacclimatized yeasts,” BioResources 9(4), 7666-7680. DOI: 10.15376/biores.9.4.76667680
Das, S. P., Gupta, A., Das, D., and Goyal, A. (2016). “Enhanced bioethanol production
from water hyacinth (Eichhornia crassipes) by statistical optimization of
fermentation process parameters using Taguchi orthogonal array design,”
International Biodeterioration & Biodegradation 109, 174-184. DOI:
10.1016/j.ibiod.2016.01.008
Das, S., Bhattacharya, A., Haldar, S., Ganguly, A., Gu, S., Ting, Y. P., and Chatterjee, P.
K. (2015). “Optimization of enzymatic saccharification of water hyacinth biomass forbio-ethanol: Comparison between artificial neural network and response surface
methodology,” Sustainable Materials and Technologies 3, 17-28. DOI:
10.1016/j.susmat.2015.01.001
de Souza Moretti, M. M., Bocchini-Martins, D. A., Nunes, C. D. C. C., Villena, M. A.,
Perrone, O. M., da Silva, R., Boscolo, M., and Gomes, E. (2014). “Pretreatment ofsugarcane bagasse with microwaves irradiation and its effects on the structure and on
enzymatic hydrolysis “Applied Energy 122, 189-195. DOI:
10.1016/j.apenergy.2014.02.020

Rezania et al. (2019). “Pretreatment of water hyacinth,” BioResources 14(1), 592-606.

603

PEER-REVIEWED ARTICLE

bioresources.com

Di Blasi, C., Signorelli, G., Di Russo, C., and Rea, G. (1999). “Product distribution frompyrolysis of wood and agricultural residues,” Industrial & Engineering Chemistry
Research 38(6), 2216-2224. DOI: 10.1021/ie980711u
Dionisi, D., Anderson, J. A., Aulenta, F., McCue, A., and Paton, G. (2015). “Thepotential of microbial processes for lignocellulosic biomass conversion to ethanol: A
review,” Journal of Chemical Technology and Biotechnology 90(3), 366-383. DOI:
10.1002/jctb.4544
Elgharbawy, A. A., Alam, M. Z., Moniruzzaman, M., and Goto, M. (2016). “Ionic liquid
pretreatment as emerging approaches for enhanced enzymatic hydrolysis of
lignocellulosic biomass,” Biochemical Engineering Journal 109, 252-267. DOI:
10.1016/j.bej.2016.01.021
Eshtiaghi, M. N., Yoswathana, N., Kuldiloke, J., and Ebadi, A. G. (2012). “Preliminary
study for bioconversion of water hyacinth (Eichhornia crassipes) to bioethanol,”African Journal of Biotechnology 11(21), 4921-4928.
Fileto-Pérez, H. A., Rutiaga-Quiñones, J. G., Aguilar-González, C. N., Paéz, J. B., López,J., and Rutiaga-Quiñones, O. M. (2013). “Evaluation of Eichhornia crassipes as analternative raw material for reducing sugars production,” BioResources 8(4), 53405348. DOI: 10.15376/biores.8.4.5340-5348
Ganguly, A., Halder, S., Laha, A., Saha, N., Chatterjee, P. K., and Dey, A. (2013).
“Effect of alkali pretreatment on water hyacinth biomass for production of ethanol,”
Advanced Chemical Engineering Research 2(2), 40-44.
Gao, J., Chen, L., Yan, Z., and Wang, L. (2013a). “Effect of ionic liquid pretreatment on
the composition, structure and biogas production of water hyacinth (Eichhornia
crassipes),” Bioresource Technology 132, 361-364. DOI:
10.1016/j.biortech.2012.10.136
Gao, J., Chen, L., Yuan, K., Huang, H., and Yan, Z. (2013b). “Ionic liquid pretreatment
to enhance the anaerobic digestion of lignocellulosic biomass,” Bioresource
Technology 150, 352-358. DOI: 10.1016/j.biortech.2013.10.026
Guragain, Y. N., De Coninck, J., Husson, F., Durand, A., and Rakshit, S. K. (2011).
“Comparison of some new pretreatment methods for second generation bioethanol
production from wheat straw and water hyacinth,” Bioresource Technology 102(6),4416-4424. DOI: 10.1016/j.biortech.2010.11.125
Harun, M. Y., Radiah, A. D., Abidin, Z. Z., and Yunus, R. (2011). “Effect of physicalpretreatment on dilute acid hydrolysis of water hyacinth (Eichhornia crassipes),”
Bioresource Technology 102(8), 5193-5199. DOI: 10.1016/j.biortech.2011.02.001
Hu, Z., and Wen, Z. (2008). “Enhancing enzymatic digestibility of switchgrass by
microwave-assisted alkali pretreatment,” Biochemical Engineering Journal 38(3),369-378. DOI: 10.1016/j.bej.2007.08.001
Imman, S., Arnthong, J., Burapatana, V., Champreda, V., and Laosiripojana, N. (2015).
“Fractionation of rice straw by a single-step solvothermal process: Effects of solvents,acid promoters, and microwave treatment,” Renewable Energy 83, 663-673. DOI:
10.1016/j.renene.2015.04.062
Jambo, S. A., Abdulla, R., Azhar, S. H. M., Marbawi, H., Gansau, J. A., and Ravindra, P.
(2016). “A review on third generation bioethanol feedstock,” Renewable &Sustainable Energy Reviews 65, 756-769. DOI: 10.1016/j.rser.2016.07.064
Mandade, P., Bakshi, B. R., and Yadav, G. D. (2016). “Ethanol from Indian agroindustrial lignocellulosic biomass: An energy evaluation,” Clean Technologies andEnvironmental Policy 18(8), 2625-2634. DOI: 10.1007/s10098-016-1179-yRezania et al. (2019). “Pretreatment of water hyacinth,” BioResources 14(1), 592-606.

604

PEER-REVIEWED ARTICLE

bioresources.com

Manivannan, A., and Narendhirakannan, R. T. (2015). Bioethanol production fromaquatic weed water hyacinth (Eichhornia crassipes) by yeast fermentation,” Waste
and Biomass Valorization 6(2), 209-216. DOI: 10.1007/s12649-015-9347-6
Miller, G. L. (1959). “Use of dinitrosalicylic acid reagent for determination of reducing
sugar,” Analytical Chemistry 31(3), 426-428.
Mukhopadhyay, S. B., and Chatterjee, N. C. (2010). “Bioconversion of water hyacinthhydrolysate into ethanol,” BioResources 5(2), 1301-1310. DOI:
10.15376/biores.5.2.1301-1310
Narra, M., Divecha, J., Shah, D., Balasubramanian, V., Vyas, B., Harijan, M., andMacwan, K. (2017). “Cellulase production, simultaneous saccharification andfermentation in a single vessel: A new approach for production of bio-ethanol frommild alkali pre-treated water hyacinth,” Journal of Environmental Chemical
Engineering 5(3), 2176-2181. DOI: 10.1016/j.jece.2017.04.043
Nigam, J. N. (2002). “Bioconversion of water-hyacinth (Eichhornia crassipes)
hemicellulose acid hydrolysate to motor fuel ethanol by xylose–fermenting yeast,”
Journal of Biotechnology 97(2), 107-116. DOI: 10.1016/S0168-1656(02)00013-5
Reales-Alfaro, J. G., Trujillo-Daza, L. T., Arzuaga-Lindado, G., Castaño-Peláez, H. I.,
and Polo-Córdoba, Á. D. (2013). “Acid hydrolysis of water hyacinth to obtainfermentable sugars,” CT&F-Ciencia, Tecnología y Futuro 5(2), 101-111.
Rezania, S., Din, M. F. M., Kamaruddin, S. F., Taib, S. M., Singh, L., Yong, E. L., andDahalan, F. A. (2016). “Evaluation of water hyacinth (Eichhornia crassipes) as apotential raw material source for briquette production,” Energy 111, 768-773. DOI:
10.1016/j.energy.2016.06.026
Rezania, S., Din, M. F. M., Mohamad, S. E., Sohaili, J., Taib, S. M., Yusof, M. B. M.,
and Ahsan, A. (2017). “Review on pretreatment methods and ethanol production from
cellulosic water hyacinth,” BioResources 12(1), 2108-2124. DOI:
10.15376/biores.12.1.2108-2124
Rezania, S., Din, M. F. M., Taib, S. M., Mohamad, S. E., Dahalan, F. A., Kamyab, H.,
and Ebrahimi, S. S. (2018). “Ethanol production from water hyacinth (Eichhornia
crassipes) using various types of enhancers based on the consumable sugars,” Waste
and Biomass Valorization 9(6), 939-946. DOI: 10.1007/s12649-017-9883-3
Rezania, S., Ponraj, M., Din, M. F. M., Songip, A. R., Sairan, F. M., and Chelliapan, S.
(2015). “The diverse applications of water hyacinth with main focus on sustainableenergy and production for new era: An overview,” Renewable & Sustainable Energy
Reviews 41, 943-954. DOI: 10.1016/j.rser.2014.09.006
Ruan, T., Zeng, R., Yin, X. Y., Zhang, S. X., and Yang, Z. H. (2016). “Water hyacinth(Eichhornia crassipes) biomass as a biofuel feedstock by enzymatic hydrolysis,”
BioResources 11(1), 2372-2380. DOI: 10.15376/biores.11.1.2372-2380
Sarto, S., Hildayati, R., and Syaichurrozi, I. (2019). “Effect of chemical pretreatmentusing sulfuric acid on biogas production from water hyacinth and kinetics,”
Renewable Energy 132, 335-350. DOI: 10.1016/j.renene.2018.07.121
Singh, A., and Bishnoi, N. R. (2012). “Optimization of ethanol production frommicrowave alkali pretreated rice straw using statistical experimental designs by
Saccharomyces cerevisiae,” Industrial Crops and Products 37(1), 334-341. DOI:
10.1016/j.indcrop.2011.12.033
Singh, A., and Bishnoi, N. R. (2013). “Comparative study of various pretreatment
techniques for ethanol production from water hyacinth,” Industrial Crops andProducts 44, 283-289. DOI: 10.1016/j.indcrop.2012.11.026
Rezania et al. (2019). “Pretreatment of water hyacinth,” BioResources 14(1), 592-606.

605

PEER-REVIEWED ARTICLE

bioresources.com

Singh, J., Suhag, M., and Dhaka, A. (2015). “Augmented digestion of lignocellulose bysteam explosion, acid and alkaline pretreatment methods: A review,” Carbohydrate
Polymers 117, 624-631. DOI: 10.1016/j.carbpol.2014.10.012
Singh, J., Sharma, R., Ghosh, P., Kumar, A., and Khan, M. L. (2018). “Imidazolium
based ionic liquids: A promising green solvent for water hyacinth biomass
deconstruction,” Frontiers in Chemistry 6, 548. DOI: 10.3389/fchem.2018.00548
Srivastava, N., Rawat, R., Singh Oberoi, H., and Ramteke, P. W. (2015). “A review onfuel ethanol production from lignocellulosic biomass,” International Journal ofGreen Energy 12(9), 949-960. DOI:
Sun, S., Sun, S., Cao, X., and Sun, R. (2016). “The role of pretreatment in improving the
enzymatic hydrolysis of lignocellulosic materials,” Bioresource Technology 199, 4958. DOI: 10.1016/j.biortech.2015.08.061
Tye, Y. Y., Lee, K. T., Abdullah, W. N. W., and Leh, C. P. (2016). “The world
availability of non-wood lignocellulosic biomass for the production of cellulosic
ethanol and potential pretreatments for the enhancement of enzymaticsaccharification,” Renewable & Sustainable Energy Reviews 60, 155-172. DOI:
10.1016/j.rser.2016.01.072
Xia, A., Cheng, J., Song, W., Yu, C., Zhou, J., and Cen, K. (2013). “Enhancing
enzymatic saccharification of water hyacinth through microwave heating with diluteacid pretreatment for biomass energy utilization,” Energy 61, 158-166. DOI:
10.1016/j.energy.2013.09.019
Xu, F., Chen, L., Wang, A., and Yan, Z. (2016). “Influence of surfactant-free ionic liquid
microemulsions pretreatment on the composition, structure and enzymatic hydrolysis
of water hyacinth,” Bioresource Technology 208, 19-23. DOI:
10.1016/j.biortech.2016.02.027
Zhang, Q., Wei, Y., Han, H., and Weng, C. (2018). “Enhancing bioethanol production
from water hyacinth by new combined pretreatment methods, “Bioresource
Technology 251, 358-363. DOI: 10.1016/j.biortech.2017.12.085
Zhang, Q., Weng, C., Huang, H., Achal, V., and Wang, D. (2015). “Optimization ofbioethanol production using whole plant of water hyacinth as substrate insimultaneous saccharification and fermentation process,” Frontiers in Microbiology
6, 1411-1419. DOI: 10.3389/fmicb.2015.01411
Zheng, Y., Zhao, J., Xu, F., and Li, Y. (2014). “Pretreatment of lignocellulosic biomass
for enhanced biogas production,” Progress in Energy and Combustion Science 42,
35-53. DOI: 10.1016/j.pecs.2014.01.001
Article submitted: August 9, 2018; Peer review completed: November 18, 2018; Revised
version received and accepted: November 23, 2018; Published: November 30, 2018.
DOI: 10.15376/biores.14.1.592-606

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