The Analytica Societas Journals

Comparative Analysis on the Production of Ethanol through Fermentation Using Different Parts of
Water Hyacinth (Eichhornia crassipes)
2
Barbaza, Marjette Ylreb, 2 Malolot, Ryan Dave, 2Dela Paz, Xech Rafael Aldrei U., 1De Castro-Cruz Kathlia*

School of Chemical Engineering and Chemistry, Mapua Institute of Technology, Muralla St., Intramuros, Manila 1002, Philippines
2
Student*, marjette.barbaza@gmail.com

ARTICLE INFO ABSTRACT

Article History: Water hyacinth is a lignocellulosic material that has potential to produce to ethanol
Volume No. 2 because of its properties. The cellulose, as a carbohydrate, can be converted to ethanol
Issue No. 8 by means of fermentation. Water hyacinth is a common aquatic plant whose abundance
Date of Submission: 16 September 2017 causes severe problems to the bodies of water. The purpose of this study was mainly
Date of Acceptance: 16 September 2017 to make use of the abundance of water hyacinth and possibly eliminate the problem
that it causes to the community. The study also aimed to utilize the different parts of
water hyacinth in the fermentation process and asses which part will have the best
ethanol yield. The study focused on identification and quantification and was
conducted by preparing the water hyacinth, by preparing the acid hydrolysate, and by
allowing the fermentation process to occur. Due to time constraints, quantification was
not carried out and only IR spectra were obtained as the result of the experiment,

Keywords: water hyacinth, ethanol, fermentation, yeast

INTRODUCTION

A lignocellulosic material can be used as source of
ethanol, an alternate to fossil fuel. Commercial ethanol is
produced by both synthetic and biological method. Cellulose is
a complex carbohydrate, or polysaccharide, consisting of 3,000
or more glucose units and is the most abundant of all naturally
occurring organic compounds. (Encyclopædia Britannica,
2016)
Water hyacinth (WH) is a type of lignocellulosic
material that is potentially available in the tropical region of the
world. WH has high carbohydrates (cellulose) and low lignin
content, impressive growth rate and no competition on land use
is a suitable lignocellulosic material for bioenergy generation.
The Water hyacinth that the researchers were able to
identify the scientific name of plant. The scientific name is
Eicchornia Crassipes. It was identified by National Museum of
the Philippines Botany Section.

Water hyacinth (WH) is a type of lignocellulosic

material that is potentially available in the tropical region of the
world. WH has high carbohydrates (cellulose) and low lignin
Figure 1: Structure of Cellulose content, impressive growth rate and no competition on land use

is a suitable lignocellulosic material for bioenergy generation.
(Shahabaldin Rezania, 2017)
Figure 2: Water Hyacinth (Eicchornia Crassipes) gathered by
researchers
As reported by (Kumar, 2009), WH contains 18–35%
cellulose, 18–49% hemicellulose and 3.5–9% of lignin
composition, which is good for extracting fermentable sugars
using different pretreatments.
Lignocellulosic hydrolysates contain substances that
inhibit microbial fermentation to desirable products like; Mono-
aromatic inhibitors which include phenolic compounds formed
during the pretreatment process by degradation of lignin
The ethanol can be produced by fermentation of sugars
extracted mostly from lignocellulosic materials.
Lignocellulosic biomass comprises of cellulose, hemicellulose
and lignin.
Fermentation is a metabolic process in which an
organism converts a carbohydrate, such as starch or a sugar, into
an alcohol or an acid. For example, yeast performs fermentation
to obtain energy by converting sugar into alcohol. (Anne Marie
Helmenstine, 2017)
Figure 3: Anaerobic Fermentation process
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This study is significant because it can contribute to
the expanse known about the water hyacinth and the production
of ethanol. This can help boost awareness on the possible uses
of water hyacinth although their presence is sometimes taken
negatively.
The scope of the study is only the determination of the
ability of the different parts of water hyacinth to produce
ethanol and the quantification of the ethanol produced using
each part. This is to compare which part produced the best
amount of ethanol. Due to time constraints, however, the
ethanol produced in the study was not quantified.
MATERIALS AND METHODS
Chemical Regents and Apparatus
During the experiment, glassware such as Erlenmeyer
flasks, volumetric flasks (100-mL and 200-mL), beakers,
pipettes (5-mL and 10-mL), suction bulb, watch glasses, hot
plates, stirring rods, spatulas and funnels were used. Other
materials like funnel support, iron stand, and wash bottles,
corks, foil, plastic, and paper were also used. The oven, blender,
miller, and autoclave were also needed to carry out the
experiment. The following reagents, yeast, 2.5 M sulfuric acid,
0.45 M calcium hydroxide, and 5 M sodium hydroxide were
used as well.
Preparation of the Water Hyacinth
Fresh water hyacinth was collected from the Pasig
River located in Intramuros, Manila in August 2017. The
collected samples were cleansed with running tap water, then
the parts (leaves, stems, and roots) were separated from each
other. The parts were chopped into small pieces and they were
oven dried at 120 ̊ C. The dried samples were grinded using a
blender and then a miller with a mesh size of . The samples were
then stored in a closed container and kept for further usage.
Preparation of the Acid Hydrolysate
Ten grams of each of the previously stored samples
was weighed and placed in a 250-mL Erlenmeyer flask wherein
100 mL of 2.5 M sulfuric acid was added. Three sets were made
for each part of the water hyacinth, resulting in a total of nine
flasks. The flasks were labeled as L1, L2, and L3 for the leaves,
S1, S2, and S3 for the stems, and R1, R2, and R3 for the roots.
The mixtures were autoclaved at 121 ̊ C for 15 minutes. It was
then cooled down to room temperature and was filtered using
cheese cloth. Another filtration was made using Whatman filter
paper no. 1 to ensure purity. The filtrate was collected as the
hydrolysate.
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Detoxification of the Hydrolysate

For detoxification, the acid hydrolysate was then

heated to 50 ̊ C for 15 minutes in order to evaporate volatile
components. After that, 0.45 M calcium hydroxide was added
and was set aside for 30 minutes in order to detoxify the
possible harmful materials that are present. It was then filtered
again using Whatman filter paper no. 1 and 5 M sodium
hydroxide was added to adjust the pH of the solution.

Fermentation

The detoxified solution was sterilized at 121 ̊ C and 1

atm for 15 minutes. For the fermentation, the concentrations of
the yeast in each flask were varied. In the flasks labeled as 1, Figure No. 4 IR Spectrum of Leaves Sample before Treatment
2.5 grams of yeast was added, 5 grams for the flasks labeled as
2, and 7.5 grams of yeast for flask 3. The flasks were loosely
plugged with cotton and were stored in a dark place for 64 hours
to allow the fermentation process to occur. Samples were
withdrawn after the allotted time and centrifuged at 50 rpm and
the supernatants were assessed for the glucose and ethanol
content.

Evaluation

To assess the success of the study, the IR spectra of the

dried samples and the pretreated ones were obtained using ATR
accessory. The spectra of both samples were compared. The
GS-MS was also used to analyze the glucose and ethanol
content of the Figure No. 5 IR Spectrum of Stem Sample before Treatment

RESULTS AND DISCUSSION

The nature of this study is to testify the capability of

each of the components or parts of the water hyacinth to
produce ethanol via the process of fermentation. Throughout
the course of the experiment, the following results were
gathered.

The researchers employed the use of the technique of

Attenuated Total Reflectance, ATR, in the determination of the
IR spectra of the three parts – the roots, the steam, and the
leaves of the water hyacinth. Furthermore, in order to see the
changes that occurred, the researchers checked the IR of the
Figure No. 6 IR Spectrum of Roots Samples before Treatment
pretreated samples, after pretreatment, and after fermentation
process.
These IR Spectra are spectra of the three samples
before treatment. It can be observed that the peaks of the leaves
Comparing the IR spectra of the following: before
and the stem samples are similar and the peaks of the roots
pretreated, after pretreatment, and after fermentation with the
sample also has the same peaks but with different intensities.
IR spectrum of ethanol, the researchers failed to produce the
Because of this result, it was analyzed that the three samples
said volatile compound.
contain and are dominant with the same compound.

To test whether the samples produced ethanol after
fermentation, the fermented samples were also tested using IR
spectroscopy. The following figures show the IR spectra of the
samples after the fermentation process:
Figure No. 7 IR Spectrum of the Fermented Leaves Sample
Figure No. 8 IR Spectrum of the Fermented Stem Sample
Figure No. 9 IR Spectrum of the Fermented Roots Sample
It can be observed from these spectra of the fermented
samples that the peaks are the same for all. Comparing these
spectra to the IR spectrum of ethanol (See Figure 10), it can be
said that the compound that was produced after the fermentation
is not ethanol, but is a carboxylic acid based on the O-H stretch
at 3000 cm -1 and the C=O stretch at 1600 cm -1. However, it
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can also be observed that the O-H stretch obtained in the IR
spectra is too strong when compared to the normal O-H stretch,
which is medium in intensity. Because of this, the researchers
were led to the assumption that there is a possibility that ethanol
was indeed produced. The IR spectra produced can be that of
the dominant component in the samples. Still, because it there
was no test done to further confirm the presence of ethanol, the
result is interpreted as there was no ethanol produced in the
process.
Figure No. 10 IR Spectrum of Ethanol
The researchers employed the usage of ATR method due
to the following reasons: Faster sampling with no preparation,
excellent sample-to-sample reproducibility, and minimal
operator-induced variations.
ATR has advanced to become the standard FT-IR sampling
technique, providing excellent data quality combined with high
reproducibility. Most samples can be analyzed with a diamond
ATR-crystal which possesses extreme chemical and
mechanical robustness. ( Bruker Optics, 2012)
CONCLUSION
In light with the experimentation process, the
researchers failed to quantify the amount of ethanol present in
each of the water hyacinth’s component. Specifically, the roots,
the stems, and the leaves.
Based on the results of the IR spectroscopy and
because the samples can have no further analysis, no ethanol
was produced in the fermentation process due to the fact that
the researchers was not able to further test the samples.
Moreover, the researchers conclude that the overall method that
was performed by the researchers was not an effective way in
producing ethanol from water hyacinth. Furthermore, yeast is
not an effective microorganism to use for the fermentation
process. Also, the time given for the fermentation process is
not enough.
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The researchers recommend the following: Choose a

better microorganism that will readily convert cellulose to
glucose, set a longer time for the fermentation process to
proceed to completion, research on a better procedure to carry
out the fermentation process to proceed to completion.

Figure 4. Preparation for Autoclave

SUPPORTING INFORMATION

A. Sample Photos

Figure 5. Detoxification of Samples

REFERENCES
Figure 1. Water Hyacinth Collected from the Pasig
River
Bruker Optics. (2012). Retrieved from
https://www.azom.com/article.aspx?ArticleID=5958

Anne Marie Helmenstine, P. (2017, April 3). What Is

Fermentation. Retrieved from
https://www.thoughtco.com/what-is-fermentation-
60819

Encyclopædia Britannica. (2016). Retrieved from

Encyclopædia Britannica:
Figure 2. Preparation of Water Hyacinth; Drying https://www.britannica.com/science/cellulose

Kumar, P. B. (2009). Methods for pretreatment of

lignocellulosic biomass for efficient hydrolysis and
biofuel production.

Shahabaldin Rezania, e. a. (2017). Ethanol Production

from Water Hyacinth (Eichhornia crassipes) Using
Various Types of Enhancers Based
on the Consumable.

Figure 3. Preparation of Acid Hydrolysate

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