Optimal Paper Hydrolysis Methodology for Bioethanol Production

Abstract

The world has a large demand for fuel, and in particular, environmentally friendly fuels such as bioethanol. There has been a growing interest in using paper as a feedstock for bioethanol production, as this generates a two-fold effect of both removing waste from landfills and meeting global energy needs. Paper undergoes acid-based or enzymatic hydrolysis, where the cellulose polymers are broken down into sugars and fermented into ethanol. The aim of this research was to assess the optimization of both hydrolysis methods by comparing the yields of ethanol production. Standard, blank, printer paper was shredded and hydrolyzed using either acid or enzymatic hydrolysis. Samples were then fermented until the reaction was determined visually as complete in an incubator. The density and boiling point of the samples were measured to determine the amount of ethanol obtained. There were no samples that used acid hydrolysis that produced any ethanol while one of the samples from enzymatic hydrolysis produced approximately 28.16 mL of ethanol. The resulting data showed that there was no significant difference in the amount of ethanol produced from acid-based or enzymatic hydrolysis of paper feedstock.

Introduction

In 2018, the United States consumed 142.86 billion gallons of finished motor gasoline, 14.38 billion gallons of which was ethanol, which is a major component of most types of gasoline.^[1] Indeed, 98% of all gasoline in the US contains ethanol, and as the global population increases so too will the demand for fuels of similar efficiency to gasoline.^[2] Ethanol concentrations typically range from 10% to 85% (E10 to E85) and in some cases 100%.^[1] Ethanol is a compelling alternative to gasoline, as it is much safer for the environment; in fact, bioethanol, typically made from corn or sugarcane, is considered atmospheric carbon neutral in that the biomass that the ethanol is produced from absorbs more CO₂ during growth than the produced ethanol emits during combustion.^[3] Thus, biomass-based ethanol would be environmentally sustainable and theoretically renewable.
The raw material used for the conversion to ethanol is known as feedstock, the most common of which is currently corn; 95% of ethanol in the US is produced from corn sources.^[2] However, this occupies significant amounts of land that could otherwise be used for growing corn as food as opposed to fuel. Another feedstock for bioethanol production is paper, which is attractive for its prevalence and the potential use of its waste. In 2017, paper and paperboard accounted for 25% (67.01 million tons) of the total 267.8 million tons of municipal solid waste in the US.^[4] In addition, 45% of the paper that is printed in offices is thrown out by the end of the day.^[5] By reusing paper as feedstock for bioethanol production, these waste quantities could be significantly reduced. Indeed, second-generation biofuels from this lignocellulosic biomass are gaining industry traction due to the significant reduction in greenhouse gas emissions.
Thus, using paper as a feedstock for bioethanol production has many potential benefits. The use of one ton of recycled paper would save about 682.5 gallons of oil, 26,500 liters of water, and 17 trees.^[6] Ethanol could be produced using existing materials, removing the need to grow corn specifically for fuel purposes, and providing more land for agriculture. Second, ethanol produced from paper would help meet the world’s gasoline demand since it could be integrated into the gasoline used to power automobiles and other machines. Testing for the optimal paper hydrolysis method in bioethanol production could help companies as they create large scale operations for this purpose.
The focus of this research is to find the optimal hydrolysis method for bioethanol production, with paper as the feedstock, by comparing enzymatic hydrolysis against sulphuric acid (H₂SO₄) hydrolysis. During hydrolysis of paper, cellulose is broken down into sugars via reaction with water. The cellulose in the paper must be broken down into glucose before the solution can ferment because cellulose is a glucose polymer that is very big and strong with bonds between the glucose monomers that cannot be broken and fermented by yeast. The glucose released during hydrolysis must be fermented using yeast to produce ethanol and carbon dioxide. Distillation then purifies the ethanol by boiling the fermented solution at a temperature between 100℃ (212℉) and 78℃ (173℉) to induce evaporation and specific condensation in the tubing apparatus for collection of the final product. Here, it is hypothesized that acid hydrolysis would generate a higher yield than enzymatic hydrolysis, as acid hydrolysis is currently the research standard when using paper feedstock. Further, there are multiple ways of conducting enzymatic hydrolysis which could impact results between different studies and make generalizing what enzymatic hydrolysis is difficult. For example, enzymatic hydrolysis could be carried out using multiple types of enzymes or an enzyme cocktail solution.^[7][8] If different studies use different enzymes and different enzyme mixtures it would be difficult to know which enzyme is the standard when enzymatic hydrolysis is used, and if variances in data across different studies are due to different enzymes or differences in procedures or other variables. There is an increasing need for this research as the global demand for fuel, and more environmentally friendly fuel, increases.

Literature Review

Htway, Thein, and Hlaning focused on bioethanol production from office waste paper using acid hydrolysis and fermentation. Different quantities of waste paper, sulphuric acid, and yeast were used to find the optimum conditions to maximize ethanol output.
Acid hydrolysis was used as a quicker, less expensive alternative to enzymes. The feedstock was provided from offices and photocopiers, and cut into smaller pieces, resulting in a larger surface area: volume ratio for increased reaction rate. In each experiment either 5g, 10g, or 15g of the cut waste paper was used. The paper was mixed with either 100 mL or 200 mL of sulphuric acid in 250 mL Erlenmeyer flasks. The hydrolysis took place in an autoclave at 121°C.
The resulting solution was filtered to remove particulates and sodium hydroxide added to maintain a pH optimum of 4.5-5 for Saccharomyces cerevisiae yeast fermentation activity. S. cerevisiae was used in differing amounts at 30 °C for 24 hours. The optimal conditions for bioethanol production were 10g of office waste paper, hydrolysis via 100 mL of 5% H₂SO₄ at 121°C for 60 minutes, and 2g of yeast.
As with most of the studies on the topic, this study used a weak acid for the hydrolysis process of converting cellulose to glucose. However, the distillation of their product was not conducted, which would otherwise purify and facilitate further testing of the bioethanol. Another limitation was that there was no specified neutralization or pretreatment process.
To consider the individual steps of the ethanol production process, research into paper hydrolysis was evaluated by Maceiras, Alfonsin, and Poole. Similar to Htway, Thein, and Hlaing, acid hydrolysis was used. Waste office paper was collected and cut into pieces with a surface area of 0.25cm². 5g of the prepared paper was placed in a flask with varying volumes of 5% H₂SO₄. Either 100 mL, 200 mL, or 300 mL of the acid was used and the solutions were placed in an autoclave at 121°C. Unlike most studies, time was varied in addition to the amount of the acid, and the time for hydrolysis was either 30 min, 60 min, 120 min, or 180 min. The solution was then filtered, centrifuged, and the solution was neutralized to a pH between 4.5 and 5 using a 5M sodium hydroxide solution.
Fermentation took place at 30°C for 24 hours at 150 rpm orbital shaking of a mixture of the hydrolyzed solution, 15% of fermentation medium, and 10% yeast suspension. The fermented solution was then distilled twice to obtain the optimal quality of bioethanol. The researchers then tested the bioethanol for certain properties like density, acid volume, color, turbidity, and refractive index to characterize the bioethanol. They came to the conclusion that the ideal amount of acid for hydrolysis was 200 mL, while the ideal amount of time for hydrolysis was 120 minutes.
Even though there were six different samples, only one of those samples was of 100 mL with H₂SO₄ and only one sample with 300 mL of H₂SO₄. The researchers should have had multiple samples for each amount of H₂SO₄ for more consistent results. This study was similar to many other studies on the topic with the use of acid hydrolysis instead of enzymatic hydrolysis, and their process that involved pretreatment, hydrolysis, neutralization fermentation, and distillation. One difference with this study was that it changed the amount of time for hydrolysis, whereas many studies only changed the amount of acid or enzymes used in the hydrolysis process. The results from this study could be used to go into further detail about other variables that affect the production of ethanol from waste paper. Our research will focus on the difference between enzymatic hydrolysis and acid hydrolysis in the production of ethanol. Unlike this study, our research will not use time variances in the hydrolysis step. However, similar to this study, hydrolysis, neutralization, fermentation, and distillation will be used in our research.
Wang et al. conducted research in 2013 towards the enzymatic hydrolysis of waste paper in the production of bioethanol. The study used newspapers, office paper, magazines, and cardboard as their feedstock. Cellic Ctec 1 was the enzyme complex used for hydrolysis. The waste paper was blended with water at 15% (w/w) for 10 minutes. Sulphuric acid was added to keep the pH of the solution at 5.2, and the saccharification process took 72 hours at 50°C. Throughout the conversion process, the yields of different types of sugar (glucose, xylose, cellobiose, galactose, mannose, and arabinose) were measured and compared among the different paper types.
The paper feedstock was split into two categories: base cases and state-of-the-art cases. The base case was used for waste papers; prepared by being pulped, enzymatically hydrolyzed at 50°C for 72 hours, fermented at 40°C for 36 hours using Zymomonas mobilis bacteria with diammonium phosphate (DAP), corn steep liquor (CSL), and recycled water, and finally distilled to obtain 99.5% (w/w) bioethanol. Diammonium phosphate (DAP) and corn steep liquor (CSL) were added in accordance with a specific fermentation process set forth by the National Renewable Energy Laboratory (NREL).
The state-of-the-art case was used for office paper and newspaper. The office paper was shredded and heated to 100°C with low-pressure steam, at which point 5% (w/w) of H₂SO₄ was added at 220°C for 2 minutes. H₂SO₄ was added to the solution to bring the pH to 5, the solution was filtered, detoxified by overliming, and then filtered again. The solution was then hydrolyzed with the Cellic Ctec 1 liquid enzymes and fermented with diammonium phosphate (DAP), corn steep liquor (CSL), recycled water, and Zymomonas mobilis. The fermented product was then distilled to obtain bioethanol. The newspaper state-of-the-art case followed a more complex set of steps compared to the office paper state-of-the-art case. The newspaper was shredded and heated to 100°C with low-pressure steam, then hydrolyzed with calcium hydroxide (CaOH₂), neutralized, and filtered. The solution then went through the same hydrolysis, fermentation, and distillation process as the office paper state-of-the-art case to obtain bioethanol.
The researchers then conducted an economic analysis by calculating the total project cost, operating cost, and discounted cash flow. Make sure to use this waitr promo code for discounts on your favorite products. The results showed that the office paper had the highest glucose yield, followed by cardboard, newspapers, and magazines. Further, the majority of the cost for the experiments came from the biomass, and only a small cost came from the enzymes. With the comparison of bioethanol and petrol gasoline, the researchers found that all but the bioethanol produced from magazine paper was economically better than petrol pump prices.
The study was very thorough in their experimentation by adding numerous steps and processes in the production of bioethanol. However, the validity of the conclusions may not be absolute, as different protocols applied to different paper types do not ensure normalized comparison.
This study differed from many other studies because the researchers did an extensive economic analysis of the ethanol and bioethanol production process, as well as varying the types of paper feedstock used. The study also used a different type of yeast and enzyme than most studies. Most studies also only measured the yield of glucose and not the yields of any other types of sugar from the hydrolysis process.
In contrast to other studies, Kang et al. conducted bioethanol production with a mixture of pine wood chips and waste paper as feedstock, obtained from the industrial byproducts of the Rock-Tenn company and the Kraft paper mill respectively. The raw materials were analyzed for moisture, carbohydrates, and ash content. The aim was to create a method of ethanol production that was both renewable and reduced waste.
Researchers followed a series of steps that closely resemble the steps used in other studies. First, they created a pulp or a mixture of paper, pine chips, and water. The paper sludge was subjected to enzymatic hydrolysis for 48 hours at 50 degrees Celsius to make sure that as much cellulose was converted as possible. The researchers then added a yeast solution to the paper sludge and put it in an incubator shaker where it fermented at 36 degrees Celsius while it spun at 150 RPM. Samples were taken from the incubator shaker at 6, 12, 24, and 48-hour intervals and analyzed for sugar and ethanol content. Instead of fermentation-based on specific time increments, a fermentation flask at room temperature could be used, and the samples could be removed when the flask indicates the end of the fermentation process. The researchers never purified ethanol, opting to measure the ethanol content in the solution after fermentation. Distillation could be used in future research to create purer bioethanol.
This article leaves out some very vital steps that should also be performed, such as distillation and the use of the enzyme cellulase. It also presented many new ways to conduct steps. For example, the fact that there is an optimal fermentation temperature could be integrated into our research.
Byadgi and Kalburgi presented a method of production of bioethanol from waste newspaper using a blend of sulfuric acid and newspaper in a 10:1 ratio. The researchers then used yeast to ferment the solution for 36 hours creating ethanol. Finally, they distilled out the ethanol to create a pure sample. This method closely resembles the method that is used in other studies and is applicable to us although some steps could be removed and others added.
First, the researchers collected waste newspaper from households, they made sure that it had not been exposed to large amounts of dust or fungi and had been dried in sunlight and stored in sealed plastic bags, so as to prevent any confounding variables. They then took the paper and shredded it into “small” pieces and placed them in a sealed bag before they underwent pretreatment. The pretreatment of the samples started with the researchers adding a blend of sulfuric acid (H₂SO₄) into the paper water solution. This step was needed to break down the paper into cellulose chains for better hydrolysis. The hydrolysis of the pretreated substrate was not clarified in the article, but an enzyme called cellulase could be used to break down the cellulose into glucose. Next, the hydrolyzed “broth” was mixed with commercial-grade yeast where the “broth” was fermented in 34 degrees Celsius for 36 hours, creating a very dilute bioethanol. Instead of the fermentation process being based on time, a fermentation flask could be used to show the researcher(s) when the fermentation process was over. The article did not state how they converted the diluted bioethanol into a purer form, stopping after the fermentation process. Distillation could be used to distill out the ethanol using a distillation tube, because ethanol boils at a much lower temperature than water, so it would be a plausible method to purify the bioethanol “broth”.
This article gives a detailed explanation on how to convert waste newspaper into diluted bioethanol, but it would have been more helpful if the researchers had distilled out the bioethanol, or separated the substrate and ethanol in some way. However, the majority of the processes could be replicated in our research.

Methodology

The aims of this research were to compare the amount of ethanol produced from each hydrolysis method; thus the independent variable is the protocol for enzymatic and acid hydrolysis, and the dependent variable is the yield of ethanol, measured as an approximate percentage and volume. Two paper hydrolysis methods were tested for ethanol output. Cellulase enzymes and 6% sulphuric acid were used for enzymatic and acid-based hydrolysis respectively. The methodology for this research was split into five components: pretreatment, hydrolysis, fermentation, distillation, and data tests and analysis. Most of the studies reviewed above shared a similar overall process and set of steps for creating ethanol from paper.
Prior to experimental implementation, the feedstock was pretreated. Standard, blank, printer paper was used to avoid ink as a confounding variable that may chemically affect the hydrolysis process. All paper was shredded and then separated into half-pound or 227g (3s.f.) samples. All the studies listed above in the literature review either cut the paper into smaller pieces or blended it into a sludge of small paper pieces. The mass of paper used for acid and enzymatic hydrolysis differed due to substrate saturation for each specific reagent.
One acid hydrolysis sample was hydrolyzed at room temperature, but all other acid hydrolysis samples were conducted in a pressure cooker. For acid hydrolysis, 6% sulphuric acid was added in a 5:1 or 10:1 ratio to paper feedstock by weight. 6% of sulphuric acid was used in order to closely follow all the reviewed studies that used acid hydrolysis, which used 5%. The acid to paper ratios was also used in similarity to the reviewed studies, where Maceiras et al. used a 4:1 ratio, and both Htway et al. and Byadgi et al. used a 10:1 ratio. The pressure cooker was kept around 100℃ (212℉) and ran for approximately one hour each day with a 10lb weighted steam release valve to pressurize the container. Complete hydrolysis lasted between 47.5 and 360 hours. A strainer was used to separate the remaining slurry from the liquid solution. The solution was then kept stirring and neutralized with the addition of two concentrations of sodium hydroxide: a stronger concentration that was in tablet form, and a weaker concentration that was in liquid form. The pH was monitored using a digital pH meter and sodium hydroxide was added until the pH of the solution was approximately neutral (a pH between 6 and 7) to ensure the samples were optimized for fermentation so the yeast would not be in a too acidic or too basic environment, which was between 40 and 43 grams of the tablets and between 0 and 15 mL of the liquid.
Enzymatic hydrolysis involved adding 4g of cellulase to an open container of paper feedstock and 3.6 to 6L of water to prevent rate-limited reactions. After one enzymatic hydrolysis sample hydrolyzed under a heat lamp, hydrolysis was later conducted in an incubator to ensure a uniformly-heated environment below 40℃ for optimal yeast function, as seen in the protocols from the literature review. The switch from the heat lamp to the incubator occurred because the heat lamp only heated the top area of the hydrolyzing solution, whereas the incubator provided even heating for the entire solution. Hydrolysis lasted between 120 and 408 hours to let the cellulase break the cellulose down into sugar. Since enzymatic hydrolysis did not involve any acids, there was no need to neutralize the solution. Once enzymatic hydrolysis ended, the paper was strained out of the sample and the liquid was added to the flask for fermentation.
The fermentation process was the same for both acid and enzymatic hydrolysis samples and took place in an incubator which was set below 40℃. Fermentation was done in an incubator which was set below 40℃ in accordance with Wang et al., Kang et al., and Byadgi et al. For fermentation, a minimum of 5g of standard baker’s yeast was added to the solution and stopped with an S-shaped twin bubble airlock stopper (shown in Figure 1), which was filled with water to observe the production of CO₂ gas as confirmation of successful fermentation. The minimum amount of time for fermentation was seven days, during which the beaker would be gently shaken two or three times a day to accelerate the process and ensure that the yeast was uniformly distributed within the solution. If visual signs of fermentation persisted after seven days, fermentation continued until visual signs ceased.

Figure 1: Diagram of an S-shaped twin bubble airlock stopper.

After completion of fermentation, a hydrometer was used to test the presence of ethanol before distillation. Distillation was also used by Maceiras et al. and Wang et al. The temperature of the mixture was kept between the boiling points of pure ethanol and pure water (78.4℃ (3s.f.) and 100℃ respectively) to ensure separation of the liquids. During the distillation process, ethanol (and some water) would evaporate, travel up to where it met the cool distillation tubing, condensed back into a liquid, and traveled into a small graduated cylinder or small beaker for collection. Through a series of pre-experimental tests to understand more about distillation, it was discovered that when ethanol evaporated, the liquid would condense into smooth streaks. Whereas when water evaporated, the vapor would condense into individual droplets which would fuse and remain stationary until a threshold maximal volume was reached, before dropping into the graduated cylinder. Typically, if there was any trace of ethanol in the sample, then the ethanol would be distilled out within the first hour of distillation. The sample was discarded if no ethanol was being distilled out, as confirmed by distillation only occurring near a temperature of 100℃, and by the resulting liquid not burning upon exposure to a flame. When a sample of liquid was collected and showed signs of ethanol content, it was then subjected to a series of distillations to obtain a maximal percentage of ethanol, typically two to three times.
The final product was then collected and tested to measure boiling points and density to characterize the proportion of ethanol in the final product. The temperature of the liquid was measured in intervals as different temperatures can alter the density of the sample. After this, the temperature at which the sample started to boil was recorded, to ensure that the density measure was more accurate due to the volume of ethanol being retained. Once all the final products were collected and analyzed, a Mann-Whitney significance test was used to compare the two hydrolysis methods to see which method produced a greater yield of ethanol.

Results

For this study, the amount of ethanol produced using acid and enzymatic hydrolysis was tested and compared. Two samples of acid hydrolysis and three samples of enzymatic hydrolysis were completed. The results of the bioethanol production experiments using both acid and enzymatic hydrolysis are shown in Figure 2. Both samples from acid hydrolysis produced no ethanol. This conclusion was reached during distillation when no liquid would distill out of the round bottom flask at temperatures under 210℉. Any liquid that did distill out did so approximately at the boiling temperature of water (212℉ or 100℃) and was not flammable. Also, any liquid vapors that condensed in the distillation tubing did not have the long, smooth stroke appearance characteristic of ethanol. Because of those factors, there was no need to test the density or boiling point of those samples since there was approximately no ethanol in those samples. Two of the three samples of enzymatic hydrolysis had the same results as those of acid hydrolysis. The last sample of enzymatic hydrolysis did produce a final product for testing.

Acid hydrolysis (ethanol yield in mL)	Enzymatic hydrolysis (ethanol yield in mL)
0	0
0	0
	28.16*

Figure 2: Results of bioethanol production experiments. The left column shows the amount of ethanol in millilitres from bioethanol production using acid hydrolysis. The right column represents the amount of ethanol in millilitres from bioethanol production using enzymatic hydrolysis.

The density of the third sample of enzymatic hydrolysis was measured. The temperature of the liquid when the density was measured was 66.4℉ (19.1℃), the mass of the sample was 30 grams, and the volume was approximately 35.2 mL. The density was calculated to be .852. To get an approximate ethanol percentage of the liquid, the density combined with the temperature was used in Figure 3. Using Figure 3, it was shown that the sample temperature was closest to 20℃ and the density was between 0.843 and 0.868. The density test claims that the sample was between 70% and 80% ethanol by weight.

Ethanol weight (%)	Temperature (°C)
	10	15	20	25	30	35	40
0	1.000	0.999	0.998	0.997	0.996	0.994	0.992
10	0.984	0.983	0.982	0.980	0.979	0.977	0.975
20	0.972	0.971	0.969	0.966	0.964	0.961	0.959
30	0.960	0.957	0.954	0.951	0.947	0.944	0.941
40	0.942	0.939	0.935	0.931	0.928	0.924	0.920
50	0.922	0.918	0.914	0.910	0.906	0.902	0.897
60	0.899	0.895	0.891	0.887	0.883	0.878	0.874
70	0.876	0.872	0.868	0.863	0.859	0.855	0.850
80	0.852	0.848	0.843	0.839	0.835	0.830	0.826
90	0.827	0.822	0.818	0.814	0.809	0.805	0.800
100	0.798	0.794	0.789	0.785	0.781	0.776	0.772

Figure 3: Densities of ethanol and water mixtures from samples derived from enzymatic hydrolysis. The far-left column represents the percent ethanol weight. The top row represents the temperature of the mixture in degrees Celsius.

28.16 is an approximation based on the density and boiling point tests. The density test suggested that the sample was between 70% and 80% ethanol. The boiling point test suggested that the sample was between 80% and 90% ethanol. In taking the average of the two predicted percentages from the two tests, we claim that the sample is approximately 80% ethanol. Since the volume of the sample was approximately 35.2 mL, 80% of 35.2 is 28.16.
A boiling point test was also conducted. The red dot in Figure 4 represents the boiling point of the third enzymatic hydrolysis sample. Using Figure 4, it was shown that the sample was between 80% and 90% ethanol by volume based on the boiling point test.

Figure 4: Boiling point of ethanol and water mixtures. The bottom x-axis represents the percent ethanol by volume of the liquid. The y-axis represents the boiling temperature of the liquid in both degrees Celsius and Fahrenheit.

After all the samples were collected, a Mann-Whitney U test was used to evaluate the hypothesis of this research. This test was used because of the small sample size (<5) which meant that population normality could not be assumed. The null hypothesis was that there was no difference in ethanol outputs between bioethanol production from acid hydrolysis and enzymatic hydrolysis. The alternative hypothesis was that bioethanol production using acid hydrolysis produced more ethanol than using enzymatic hydrolysis. A right-tailed test was used with an alpha value of 0.05. When using the data from this research in the Mann-Whitney U significance test proceed with caution because the sample sizes of the two hydrolysis methods were less than 5. The results of the significance test are shown in Figure 5. Since the p-value was greater than , the null hypothesis was accepted; there was sufficient evidence to suggest that there was no significant difference in ethanol output between acid and enzymatic hydrolysis.

Mann-Whitney U significance test data

Null hypothesis: Alternative hypothesis: proceed with caution due to the small sample size. proceed with caution due to the small sample size. U = 2 Test statistic Z = -1.224745 P-value = 0.889664 Result 0.889664 > 0.05, fail to reject

Figure 5: Data for the Mann-Whitney U significance test and results of the significance test.

Discussion

The limited sample size may be due to the difficulties in maintaining specific conditions; in early testing, the toxicity of ethanol rose during fermentation until the yeast died and the fermentation process ceased, thus limiting the amount of sugar converted to ethanol. Furthermore, there was only one sample that had traceable ethanol content, which could potentially represent an error with the procedure.
This research is merely applicable for optimal bioethanol production from paper using either enzymatic or acid hydrolysis. This research is not applicable for the optimal amount of enzymes or acid needed for hydrolysis, fermentation time, distillation time, yeast for fermentation, paper to acid or paper to enzyme ratio, or optimal temperature during hydrolysis or fermentation.
This research could be expanded by determining parameters such as the optimal mass of enzymes or volume of acid necessary for hydrolysis, experimental conditions, species of yeast used, or feedstock: reagent ratio.
The major limitation to this study was time. At the beginning of this research, there was an expected four and a half months to collect data, which ended prematurely due to the COVID-19 pandemic. The exclusion of these samples potentially skewed the results of this research. Further, the amount and type of equipment were restricted; some equipment used in similar studies, such as autoclaves, mediums for cellulase enzymes, and refractometers, were not available for use in this study.
The hydrometer created confusion within the study that could be better evaluated in future research. Every time the hydrometer was used after a sample finished fermenting, it showed that there was no ethanol in the sample. However, when the hydrometer was tested on other small scale side tests it was working correctly. Even with the negative results from the hydrometer, samples still went through distillation. The false negatives of the hydrometer made it confusing for when fermentation was actually completed and if there was actually ethanol present in the sample.
To improve the procedure of this study it would be beneficial to calculate the sugar content after hydrolysis for both methods. Calculating the sugar content after hydrolysis would show which method produced more sugar, and thus theoretically, more ethanol. The hydrolysis methods could then be compared to determine which produced more sugar during hydrolysis and which produced more ethanol. Theoretically, the hydrolysis method that produces the most sugar should produce the most ethanol. If that does not happen, that could be an opportunity to investigate other factors that may influence the amount of ethanol produced after the hydrolysis stage. There are two ways to measure the sugar content of a solution; a refractometer could be used to measure the °Bx to represent sucrose content, or reducing sugars could be measured using dinitrosalicylic acid as part of the DNS method.^[9][10]
For future research, it would be advised to use small amounts of paper per sample. During this study, it was quickly observed that using half-pound sizes of paper was too large, making the process costly and difficult to maintain in the correct apparatus for each reaction. Monitoring the pH of the samples during fermentation would also be useful, as the solution becomes more acidic, killing the yeast at a pH below 4. Adding more yeast to the fermenting solution would not restimulate fermentation because of the toxicity of the solution once a certain threshold of ethanol has been created. Thus, to maintain yeast activity, a buffer such as sodium bicarbonate or potassium bicarbonate should be added to the solution Another topic that could be tested in future research would be which hydrolysis method was the simplest and least expensive. The enzymatic hydrolysis seemed much simpler because there was no need to hydrolyze in an autoclave or to neutralize the solution after hydrolysis. This could be significant in industrial applications, where large scale facilities require extra steps and expenses for autoclaves and neutralization chemicals with acid hydrolysis.

Conclusion

From the hypothesis test, it can be concluded that there is insufficient evidence to suggest that bioethanol production using acid hydrolysis produces more ethanol than using enzymatic hydrolysis. The original hypothesis that bioethanol production using acid hydrolysis produces more ethanol was not proven, but also was not disproven; previous researchers had demonstrated high yields with acid hydrolysis methods, but this was not reflected in this study. However, due to the limited sample size, the validity of these results must be further supported by increasing sample size in later experiments. The validity of these results may also be limited due to the differing feedstock concentrations since the comparison of results across different samples may become difficult. The results of this research cannot be compared to other research because there is no known research that compares bioethanol production using acid and enzymatic hydrolysis. More research would need to be conducted to verify or contradict the results of this research. While the results of this research did not provide conclusive evidence as to which hydrolysis method produces more ethanol, it could still be applicable and valuable to future researchers who want to compare hydrolysis methods for bioethanol production with paper. As the world looks for new, environmentally friendly ways to meet its fuel needs using reusable materials, finding the most effective and efficient hydrolysis method will prove invaluable to large scale productions of bioethanol with paper as the feedstock.

Acknowledgements

Special thanks to Dr. Kurt Guter for volunteering his time to assist this research with his expertise. Special thanks to Mr. Joe Rasmus for purchasing needed materials for this research and providing advice.

Appendix

A complete list of all the materials used in this research is shown below:

• Paper shredder
• Sodium hydroxide pellets and liquid for neutralization after hydrolysis
• Bunsen burner for distillation.
• 500mL round bottom boiling flask
• 150mL round bottom boiling flask
• 1,000mL round bottom flask with two necks for distillation
• Digital thermometer to measure the temperature of the liquid solution during distillation and boiling point tests
• Distillation column 500mm
• S-shaped twin bubble airlock and stopper
• Scale to weigh the paper, cellulase enzymes, and yeast
• Beakers and graduated cylinders of various sizes to measure water, sulphuric acid, sodium hydroxide, and to collect ethanol during distillation
• 2000mL beakers for fermentation
• Magnetic stirrer and various magnetic stir bars
• Digital pH meter
• Rubber gloves for safety protection
• Protective goggles for safety precautions
• Cellulase enzymes (powder)
• Sulphuric acid (6%)
• Standard blank printer paper
• Electric pressure cooker used for acid hydrolysis
• Tap water used during enzymatic hydrolysis
• Standard active dry baking yeast
• Hydrometer 0-100%
• Large Incubator
• Glass stir sticks for mixing during hydrolysis
• Large polyethylene plastic open containers for enzymatic and acid hydrolysis
• 5,000 mL glass bowl for acid hydrolysis in the pressure cooker
• Strainer to remove leftover paper sludge after hydrolysis

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13. S-shaped twin bubble airlock stopper https://i5.walmartimages.com/asr/74d5dbf2-8de2-4d38-a137-43fe94ec2bbd_1.312ff377ea6c8f76be128ca128ce6f2e.jpeg?odnWidth=612&odnHeight=612&odnBg=ffffff

Biography

Matthew Wilson

Matthew Wilson is 18 years old and a senior at Williamston High School in Williamston, Michigan. He is a member of the Math and Science Academy (MSA) which is an advanced math and science program for high school students. He is a member of the cross country and tracks and field teams. In Matthew’s spare time he likes to backpack and visit National Parks.

Jack Schafer

Jack Schafer is 17 years old and attends Williamston Highschool. Jack is a member of the Williamston Math and Science Academy where students are given opportunities such as advanced math and science classes, and the ability to take a research class in their junior year. Jack enjoys many activities from traveling to building drones and other remote control devices. Jack is also a part of the track team, the cross country team, WHS InvenTeam, Innovative vehicle design team, and class president of student government.