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 Water hyacinth is a lignocellulosic material that has potential to produce to ethanol
because of its properties. The cellulose, as a carbohydrate, can be converted to
Article History: ethanol by means of fermentation. Water hyacinth is a common aquatic plant whose
Volume No. 2 abundance causes severe problems to the bodies of water. The purpose of this study
Issue No. 8 was mainly to make use of the abundance of water hyacinth and possibly eliminate
Date of Submission: 16 September 2017 the problem that it causes to the community. The study also aimed to utilize the
Date of Acceptance: 16 September 2017 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

ABSTRACT

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 Figure 1: Structure of Cellulose
naturally occurring organic compounds. [ CITATION
Enc161 \l 13321 ]
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 content, impressive growth rate and no competition on
land use is a suitable lignocellulosic material for bioenergy
generation. [ CITATION Sha17 \l 13321 ]
Figure 2: Water Hyacinth (Eicchornia Crassipes) gathered by
researchers
As reported by [ CITATION Kum09 \l 13321 ] , 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. [CITATION Ann \l 13321 ]
Figure 3: Anaerobic Fermentation process
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.
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
pretreated samples, after pretreatment, and after fermentation
process.
Comparing the IR spectra of the following: before
pretreated, after pretreatment, and after fermentation with the
IR spectrum of ethanol, the researchers failed to produce the
said volatile compound.

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. Figure No. 5 IR Spectrum of Stem Sample before Treatment
The GS-MS was also used to analyze the glucose and ethanol
content of the

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.
Figure No. 6 IR Spectrum of Roots Samples before Treatment
These IR Spectra are spectra of the three samples
before treatment. It can be observed that the peaks of the
leaves and the stem samples are similar and the peaks of the
roots sample also has the same peaks but with different
intensities. Because of this result, it was analyzed that the
three samples 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 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.[ CITATION Bru12 \l 13321 ]
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.

The researchers recommend the following: Choose a

better microorganism that will readily convert cellulose to
Figure 3. Preparation of Acid Hydrolysate
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.

SUPPORTING INFORMATION
Figure 4. Preparation for Autoclave
A. Sample Photos

Figure 5. Detoxification of Samples

Figure 1. Water Hyacinth Collected from the Pasig
River
REFERENCES

Figure 2. Preparation of Water Hyacinth; Drying

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-
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Encyclopædia Britannica. (2016). Retrieved from Encyclopædia Britannica: 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.