The Extraction of Bioethanol from Pineapple (Ananas comosus) Peelings Through Simultaneous Saccharification and Fermentation Using the Yeast Saccharomyces cerevisiae

AN INVESTIGATORY PROJECT
SUBMITTED AS AN ENTRY TO THE
16TH INTERNATIONAL ENVIRONMENTAL PROJECT OLYMPIAD (16TH INEPO)
16. INEPO ÇEVRE PROJE OLİMPİYADI

FATİH KOLEJİ
(FATIH COLLEGE)
ISTANBUL, TURKEY
1-4 JUNE 2008

Avril Rodiel Bries

Quezon City Science High School (Regional Science High School for NCR)
REPUBLIC OF THE PHILIPPINES- CUMHURIYET FİLİPİNLER
School Year 2007-2008

Mr. Noel Pablo Diaz
Research Adviser

Abstract:

On average, 435,000 metric tons of pineapples are produced annually in the Philippines, which is one of the country’s leading commercial fruit products. However, there are a lot of unused excess parts of the pineapple, notably the peelings, which are considered as waste and contribute to the country’s garbage problem.

There is a global oil crisis, as the demand for petroleum increases each year while our supplies are rapidly being depleted. Bioethanol, a principle biofuel, is a natural alternative to gasoline.

One solution to both dilemmas is to produce bioethanol from pineapple plant peelings. This will be made possible by extracting the glucose content of the peelings and fermenting it through the process of Simultaneous Saccharification and Fermentation (SSF) using the yeast sample. Peel samples, coming from Ananas comosus, will undergo a series of physical and chemical analyses to determine the glucose content, which will be utilized to yield bioethanol. The SSF process will be manipulated in terms of fermentation time and buffer level used to determine the best variables for biofuel production.

Introduction

A. Background of the Study

The concept of producing bioethanol from pineapple (Anae ananas comosus) peelings came from the notion that pineapple peelings have considerable amounts of glucose, of the oligosaccharide group. Included in this group are d-glucosamine, d-mannose, d-xylose, l-fucose in ratios of 2:2:1:1. Exhaustive proteolysis resulted in the isolation of bromelains, glycopeptides containing only Asx, Glx and Ser. Periodate oxidation, methylation, and glycosidase digestion showed that the oligosaccharide chain has a highly branched structure in which all the neutral sugars are in non-reducing terminal positions and both N-acetyl-d-glucosamine residues occur in internal positions. This proves that pineapple peelings can be a potential source of bioethanol since the sugar found in its cellulosic material is the chief raw material of bioethanol production.

Moreover, a recent study entitled “Ethanol production from alfalfa fiber fractions” shows that Simultaneous Saccharification and Fermentation produced the greatest yield of ethanol from Alfalfa fiber fractions. Results further show that said Simultaneous Saccharification and Fermentation is the best method to be used for extracting bioethanol.

This study looks into the feasibility of producing greater yield of ethanol from pineapple peelings using the SSF process. While preliminary studies have been conducted researching the extraction of bioethanol from various root crops, cellulosic material and algae, none have specifically targeted pineapple peelings as a source of bioethanol. Moreover, no other studies have researched how certain variations in the buffer level and fermentation time during the SSF process affects bioethanol production from pineapple peelings.

B. Statement of the Problem

The study aims to: • Extract bioethanol from pineapple (Ananas comosus) peelings through the SSF process using the yeast Saccharomyces cerevisiae (Baker’s yeast).

Specifically, the study attempts: • To determine whether or not the pineapple peelings will be a feasible source of bioethanol • To know whether variations in the SSF process in terms of the buffer levels and length of the fermentation time will have a significant effect on the net bioethanol yield

C. Hypothesis If the pineapple’s (Ananas comosus) peelings are subjected to the SSF process using the yeast Saccharomyces cerevisiae, then a significant amount of bioethanol can be extracted. • There is a significant difference in the average amount of ethanol produced when fermentations times are varied. • There is a significant difference in the average amount of ethanol produced when different amounts of buffer are used. • There is an interaction between the varied fermentation times and the amounts of buffer used. • There is a significant difference in the percentage yield of bioethanol that uses SSF process and the yield of that which does not.

D. Significance of the Study The world is currently undergoing an oil crisis. Due to our continuous annual oil consumption, our reserves are quickly being depleted, with scientists predicting that at our current rate of consumption, in just 40 years, our entire fossil fuel supply will run completely out. However, oil consumption rates are increasing rather than decreasing, thereby using up even more of this finite resource.

Moreover, while oil has a number of uses in the global community, burning it increases the trace gas concentration in the atmosphere and causes significant environmental problems such as global warming.

Further, the Philippines is experiencing a problem in waste management. Millions of tons of garbage are being thrown away by Filipinos each year, and the accumulated waste takes up valuable real estate and is a source of land pollution.

The pineapple (Ananas comosus) is one of the leading produce in the Philippines, as it thrives in tropical climates. However, the pineapple’s peelings have no commercial value, and are therefore merely thrown away, contributing to the Philippines’s waste problem.

This study aims to utilize pineapple peelings as a raw material to obtain bioethanol, thereby significantly contributing to the reduction of both the Philippines’s total waste count and the growing global oil crisis.

E. Scope and Limitations

The study was guided by the following scope and limitations: 1. Production of bioethanol from Ananas comosus peelings was conducted to determine the peelings’ potential as a bioethanol source. 2. The peel samples were collected and forwarded to the Philippine Institute of Pure and Applied Chemistry (PIPAC) in the Ateneo de Manila University Campus, Loyola Heights, Quezon City, for Total Sugar Determination. 3. The chemicals and other laboratory materials were obtained from the National Institute of Microbiology and Biotechnology (NIMBB). Laboratory preparations and the Simultaneous Saccharification and Fermentation (SSF) of the peelings were conducted at the Fermentation Engineering and Service Laboratory (FESL), Enzyme Laboratory, and the Philippine National Microbial Collection (PNCM) in the University of the Philippines-Los Banos, Laguna (UPLB). The subsequent chemical analysis of the bioethanol produced was conducted at the Institute of Chemistry, University of the Philippines, Diliman, Quezon City. 4. The attempt to use the peelings to conduct bioethanol production experiments, testing of combinations and refinement of the different aspects of the methodology for optimal yield, as well as the feasibility analyses and studies about the economic technicalities of the research project were not included because of the unavailability of necessary protocols for fermentation methodologies for the said samples, as well as the inadequacy of time, finances and technical expertise of the researchers.

Methodology

Collection, Preparation, and Total Sugar Determination of Pineapple Peelings
Waste peelings were collectedfrom pineapples.These were then ground into pulp using a blender, placed in a sterile container, and stored for the subsequent sugar concentration analysis.
The container was then labeled for identification in preparation for the Total Sugar Determination. Fifty grams (50g) of the pulp was subjected to automatic chemical analysis of its total sugar content. The test determined that the glucose content of the pineapple peelings was suitable for saccharification and fermentation.
Having determined the glucose content of the peelings to be used for SSF, 120g of peelings were cut into smaller particles and ground into pulp for fifteen minutes using a blender to make the samples more susceptible to enzyme attack.

Preparation of the Culture Media
Two (2_ 100mL flasks of Glucose-Yeast-Peptone (GYP) medium were prepared by diluting 20 grams of glucose and 10 grams of peptone in 1L of distilled water and sterilized for 20 minutes at 121˚C. All glassware and laboratory apparatuses were sterilized in autoclave for one hour at 121˚C.

Preparation and Inoculation of the Saccharomyces cerevisiae
Five (5) mL suspension of Saccharomyces cerevisiae strain was obtained and inoculated into the prepared broth. This was then incubated at room temperature on a rotary shaker at 200 rpm for 24 hours before inoculation into the fermentation medium.

Preparation of Enzyme Solution
Twenty-five grams (25g) of powdered cellulase and xylanase enzymes were weighed and transferred into two separate flasks. Each was diluted with 200mL of prepared sodium hydroxide (NaOH) buffer. Each flask was vigorously shaken through mechanical means for thorough incorporation of the enzyme with the liquid buffer.

Preparation and Experimentation of the Peelings with Saccharomyces cerevisiae through Simultaneous Saccharification and Fermentation
Eight flasks were used for the experiment and labeled depending on the type of SSF manipulation they would undergo. The flasks were then divided into two groups, A and B. Each subgroup is composed of four flasks each, containing 15g of pulp per flask. Fifty (50) mL of prepared 2.0% sodium hydroxide (NaOH) buffer were distributed to each flask of group A, and 100mL of the same buffer were distrubted to each flask of group B. Groups A and B were inoculated with 5mL of cellulase and xylanase solution and 5mL suspension of yeast cells.

The first two flasks of the first group, labeled A1, were allowed to ferment for 24 hours, while the second pair of flasks from the same group, labeled A2, was allowed to ferment for 72 hours.
Likewise, the first two flasks of the second group, labeled B1, were allowed to ferment for 24 hours, while the second pair of flasks of the same group, labeled B2, was allowed to ferment for 72 hours.

Bioethanol evaporation was prevented and aerobic conditions were maintained by placing cotton plugs on all flasks. After their respective fermentation times, the pulp broth was filtered through orindary filter paper, and the filtrate of each flask was immediately submitted for chemical analysis.

Chemical and Subjective Analysis of Liquid Product for Bioethanol Content through Gas Chromatography

All the filtrates of the flasks of the two groups were sent for chemical analysis to determine the bioethanol content of the liquid filtrate determined through gas chromatography. Samples of each liquid filtrate in each of the flasks were loaded to the gas chromatogram for chemical analysis, and the results of the test were recorded and analyzed.

Results and Discussion

|FERMENTATION TIME (hours) |LEVEL OF BUFFER (mL) |
| |50.0 mL |100.0 mL |
|24 |5.22% |3.37% |
| |5.21% |3.26% |
| |Mean concentration: 5.215% |Mean concentration: 3.315% |
|72 |3.63% |2.02% |
| |3.92% |1.98% |
| |Mean concentration: 3.775% |Mean concentration: 2.000% |

Based on the findings obtained in this study, the following conclusion was drawn:
It can therefore be concluded that bioethanol can be extracted though Simultaneous Saccharification and Fermentation from the peelings of pineapple (Ananas comosus) using the yeast Saccharomyces cerevisiae. The highest concentration of bioethanol yield was obtained from Flask A1 (with mean bioethanol concentration of 5.215%). The lowest concentration of bioethanol yield was obtained from flask B2 (with mean bioethanol concentration of 2.000%).

The findings also indicate that the amount of bioethanol produced in the group with the 24-hour fermentation time is greater than the bioethanol produced after 72 hours, and that the lesser buffer level used in the SSF process results in greater bioethanol yields compared to the group with higher buffer level. Moreover, based on the two-way ANOVA classification with interaction, we can conclude that the two independent variables, the fermentation time and the buffer level, have a complex interaction with each other in terms of bioethanol yield. All in all, these results state that the pineapple (Ananas comosus) peelings are a feasible potential raw material for bioethanol extraction.

Summary, Conclusion and Recommendations

The project was conducted to determine if the pineapple (Ananas comosus) peelings can yield a significant amount of bioethanol through the simultaneous saccharification and fermentation (SSF) process using the yeast Saccharomyces cerevisiae.

Different variations were manipulated to determine whether or not several factors in the saccharification and fermentation process have a significant effect on the bioethanol yield, i.e. the different buffer levels and the duration of the fermentation process. The amounts of bioethanol produced were then analyzed chemically through gas chromatography.

The findings in the gas chromatography showed that the pineapple (Ananas comosus) peelings can indeed yield significant amounts of bioethanol. The results also showed that the varying conditions in the SSF process that were manipulated in the experiment, namely the different levels of buffer and length of the fermentation process indeed has a significant effect on the amount of bioethanol produced and on the efficiency and effectivity of the whole saccharification and fermentation process.

This project entitled “The Extraction of Bioethanol from Pineapple (Ananas comosus) Peelings through Simultaneous Saccharification and Fermentation Using the Yeast Saccharification cerevisia” will indeed be a great help to the economy and the environment if ever this research becomes successful. It will benefit the economy as this will use only wasted pineapple peelings; it will not be in any way detrimental to the environment-friendly energy source and this will consequently lessen the pollution worldwide. This research will also answer the crisis of looking for a clean, alternative source of energy.

Based on the findings in this study, the following conclusion was drawn:

It can therefore be concluded that bioethanol can be extracted through Simultaneous Saccharification and Fermentation from the peelings of pineapple (Ananas comosus) using the yeast Saccharomyces cerevisiae. The highest concentration of bioethanol yield was obtained from flask A1 (with mean bioethanol concentration of 5.215%). The lowest concentration of bioethanol yield was obtained from flask B2 (with mean bioethanol concentration of 2.000%).

The findings also indicate that the amount of bioethanol produced in the group with the 24-hour fermentation time is greater than the bioethanol produced after 72 hours, and that less level of buffer used in the SSF process results to greater bioethanol yields compared to the group with higher buffer level. Moreover, based from the two-way ANOVA classification with interaction, we can conclude that the two independent variables, the fermentation time and the buffer level, have a complex interaction with each other in terms of bioethanol yield. All in all, these results state that the pineapple (Ananas comosus) peelings are a potential raw material for bioethanol extraction.

The researchers would recommend the people to not throw away their pineapple peelings whenever they consume the fruit, and also the pineapple-made product manufacturers to store the peelings of the pineapples they make use of.

The researchers would also recommend local waste management committees to religiously collect pineapple peelings from citizens and submit them to laboratories.

Lastly, the researchers would like to recommend scientists to extract bioethanol from these pineapple peelings through the SSF Process using Saccharomyces cerevisiae and to conduct the experiment by using less buffer level, no agitation, and 24-hour fermentation period. Further research regarding other combinations and refinement of the different areas of this methodology for higher bioethanol yield, as well as the feasibility examinations and further studies about the economic technicalities of the study would as well be highly recommended.

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