Presentation 

Introduction

Introduction and Background

Climate change from fossil fuels is a critical issue that requires urgent action. According to the 2021 Intergovernmental Panel on Climate Change (IPCC) report, world temperatures will rise to 1.5 ℃ above pre-industrial levels unless deep reductions to greenhouse gas emissions are made in this decade (IPCC, 2021). Due to our modern reliance on carbon-based fuels, we cannot immediately move away from them; however, supplementing dirtier fuels with high carbon emission-to-energy ratios with cleaner alternatives should allow us to lower carbon footprints significantly (Economist, 2021). Additionally, urbanization and other geopolitical changes have made fossil fuels more expensive and less accessible, with economies often ill-prepared for supply chain disruptions and fraught global relations (Economist, 2021). Alternative energy sources are a promising option for those seeking energy independence (Maity, S., & Mallick, N., 2022). 

 Among these, bioethanol stands out as a compelling alternative fuel for numerous reasons. Bioethanol produces less greenhouse gasses than gasoline: one gallon of pure ethanol produces 12.72 pounds of carbon dioxide vs. the 19.64 pounds of carbon dioxide produced by burning a gallon of unsupplemented gasoline (EIA, 2014). Furthermore, ethanol is already widely used as a fuel additive in many parts of the world. E10, a blend of approximately 10% ethanol to gasoline, is readily usable in practically all modern gasoline engines. E85 or ‘flex fuel’, a blend of 51-83% ethanol and gasoline, can be used in engines fitted with a conversion kit and is widely used in some countries such as France and Sweden (Reuters, 2021) (Epure, 2020). In Brazil, ethanol is available as a stand-alone fuel, commercially sold as E100 (100% ethanol) for flex fuel engines. Geopolitically, the only energy security concern for domestically produced bioethanol is the availability and accessibility of the feedstock used to refine it; for this reason, most production sites in the United States are located in the Midwest, close to cornfields (EIA, 2017). 

The primary feedstock for bioethanol is currently purpose-grown, sugar-rich crops; in the United States it is effectively all sweet corn (RFA, 2022). The primary process for bioethanol production is fermentation, a biological process that many organisms, such as yeast and bacteria, use to extract energy from organic compounds, creating ethanol as a by-product. Sugar monomers, such as glucose, xylose, and others, can be broken down into ethanol and carbon dioxide, generating ATP for the organism (Hossain, N. et al 2017).  Ethanol produced is then typically extracted through distillation, a three-stage process involving: 1) Solid removal with a beer column, 2) High proof ethanol distillation with a rectifying column, and 3) Low proof ethanol distillation with side stripper columns (Midwest Grain Processors, 2008). Each step takes advantage of ethanol’s lower boiling point than water; by keeping the temperature around 174 F (79 C), ethanol can boil off without allowing water to become steam (Aditiya, H.B.,  et al., 2016) (S. Azhar et al., 2017). For large-scale industrial fermentation, two prior steps are required before fermentation takes place (Chen et al., 2018). Sugar-rich crops are first milled to increase surface area before undergoing saccharification. Saccharification, also known as hydrolysis, is the process by which longer starch polymers are broken down. In modern industrial processing, this is achieved through acid hydrolysis, which uses an acid to separate sugar monomers from the polymer chain, at which point the resulting substrate is fermented (Megawati et. al., 2022) (Chen et al., 2018) (Nunes et al., 2021). However, downsides of current industrial bioethanol production methods include the requirement for large amounts of sugar-rich crops, eg., sweet corn in the United States and sugar cane in other countries, which are incentivized and supported by controversial government subsidies (Babock, 2011). Critics argue that using purpose-grown crops for fuel production alters food-fuel balances and can cause artificial price inflation, which has motivated scientists to search for alternative, next-generation production methods (Tyner, 2008). 

While today’s first-generation bioethanol production largely utilizes starchy sugars derived from sugar-rich crops, research into next-generation bioethanol production has largely been concerned with lignocellulosic biomass (Aditiya, H.B., et al., 2016). Also known as ‘dry plant matter’, lignocellulosic biomass refers to any green plant material, such as leaves, stalks, and shoots, all of which are largely made of cellulose, hemicellulose, and lignin (Wang et al., 2014). The main advantage of using lignocellulosic biomass is that it is an abundant agricultural waste product that, if widely utilized, would bypass the need for purpose-grown crops. Next-generation bioethanol production from lignocellulosic substrates involves pretreatment steps such as physical milling, ammonia fiber explosion, and steam treatment to break down the substrate into sugar polymers that can undergo saccharification (Aditiya, H. B. et al., 2016) (Yu et al., 2018). Enzymatic hydrolysis can also break down longer polysaccharides in conditions more amenable to fermenting organisms than acid hydrolysis and without risk of corrosion (Alvira, P. et al., 2010). However, pre-treatment methods require specialized facilities and have to be tailored to the cellulose, hemicellulose and lignin composition of the substrate for maximum efficiency (Tse et al.; 2021) (Mansfield et. al, 1999) (Alvira, P. et al., 2010). Many of these methods of next-generation bioethanol production from lignocellulosic biomass require further research and are not yet economically viable enough to replace widely proliferated first-generation bioethanol production (Tse et al.; 2021) (Mohd Azhar, S. et al., 2017). 

Bioethanol can also be produced from other substrates that do not require second-generation treatments and procedures, in particular, food waste products, making it a value-added source of energy, while there are also secondary environmental benefits as less waste goes to landfills (Yoo, 2021). Agricultural food wastes such as fruits, vegetables, roots, and tubers make up more than 45 percent of total food waste globally from harvest to distribution (FAO, 2019). Foodstuffs in these categories are typically rich in sugars such as sucrose, fructose, glucose and galactose as well as carbohydrates like starches and cellulose (Lin, Y. & Tanaka, S., 2006) (Rahmanto et al., 2022). Agricultural food waste that either should not or will not be consumed by humans contain unexploited energy in sugars which can be fermented into a green fuel alternative (Yoo, 2021). As one example on a small scale, a study by Ahmad et al., 2021 investigated bioethanol production using date palm fruit waste, which is the largest agricultural waste in Pakistan. Low-quality dates were fermented for 72 hours in a yeast extract medium with Saccharomyces cerevisciae, also known as common baker’s yeast. This relatively basic and inexpensive procedure yielded 15% ethanol concentration, which is a modest yet promising outcome compared to the output of other raw agricultural waste products such as corn stover (before pre-processing). The main limiting factor to using food waste products as a feedstock in industrial production is their wide variability in molecular composition, with some fermenting organisms much better suited to one type of food waste than another.  Thus, the choice of fermenting organisms is critically important as well as the development of fermenting organisms that ferment a wide array of substrates (Roukas & Kotzekidou, 2022). 

Conceptually, bioethanol production from agricultural food waste can be achieved on an industrial scale using a wide variety of fermenting organisms, each of which have specific specializations but also corresponding drawbacks. Organisms require different fermentation conditions, such as certain pH levels or specialized media (Aditiya, H.B., et al., 2016). While certain bacteria and other organisms have bioethanol producing capabilities such as Zymomonas mobilis, which has been shown to be even more efficient than some yeast strains, yeast remain popular largely due to their variety and versatility (Aditiya, H. B., et al., 2016). Yeast are able to produce bioethanol in a wide range of concentrations, pHs, and media, without the need for more specialized environments. They also have high resistance to the ethanol they produce compared to other organisms, and there are many different yeast species for researchers to choose from with varying properties (Mohd Azhar S. et al., 2017). Among these, the most ubiquitous strain is undoubtedly S. cerevisiae, which has been used for centuries to make alcoholic beverages and remains the premiere strain used in industrial production due to its generous pH tolerance, as well as its low cost and familiarity (Mohd Azhar S. et al., 2017).

Furthermore, certain strains of wild and genetically modified strains of yeast allow for a great amount of specialization and customizability. A 2017 review article by Mohd Azhar et al. details several different yeast strains with potentially useful abilities. For instance, a wild type S. cerevisiae strain, KL17, is the most efficient at producing ethanol due to  its ability to simultaneously ferment glucose and galactose, which is particularly useful for the production of ethanol from algal biomass (Kim, J.H. et al., 2014). Lab strains have also been developed for industrial production; a 2004 study from FEMS Yeast Research published by Oxford was able to create yeast strains specialized for specific environmental stressors using induced evolution techniques. By subjecting 96 plate wells to adverse conditions in multiple rounds and selecting for the most fit colonies from each round, they found the most tolerant to freezing and thawing was also highly tolerant for the other environmental stressors tested, with a 62-fold increase in ethanol resistance over the wildtype, along with a 89-fold increase in temperature resistance and a 1429-fold resistance in oxidative stress (Çakar, Z. P. et al, 2005). Additionally, recombinant DNA technology can be used to genetically modify yeast for increased resistance to ethanol, heat, and other inhibiting factors (Doğan, A. et al., 2014). However, in bioethanol research S. cerevisiae generally serves as a baseline which other organisms are compared to, which is why I will be using it in initial testing of food waste substrates.

The study I am replicating, Khoshkho et al., is an investigation into whether significant quantities of bioethanol can be derived from waste carrot pulp and molasses inoculum using S. cerevisiae. In Iran, where the researchers were based, waste carrot pulp from vegetable juicing plants and waste beet molasses from sugar beet processing are readily available, with both containing unexploited sugars after disposal, making them potential alternative feedstocks and inoculum, respectively, for bioethanol production. Carrots were juiced, and the waste pulp was collected, washed, dehydrated, and ground into a fine powder. Quantities of the carrot pulp ranging from 0 to 10 grams were fermented in bottles at 28 C over 72 hours, using dilutions of yeast and beet-molasses inoculum ranging from 0 to 25 percent. Carbon dioxide production, a waste product of ethanol fermentation, was estimated using balloons placed over the mouth of the bottles during fermentation, with larger balloons suggesting greater amounts of bioethanol production. Afterwards, the contents of each bottle were distilled to separate the ethanol from the rest of the solution, and the ethanol yield was measured. The highest ethanol yield of the experiment came from a combination of 10 grams of carrot pulp and 50 mL of diluted beet molasses and yeast inoculum yielded about 10 mL of ethanol, which the researchers consider promising.

 My study has three arms in each of which I will use S. cerevisiae to produce bioethanol in: 1) molasses inoculum with no substrate as my negative control, 2) carrot pulp with molasses inoculum as my experimental arm, and 3) cane sugar with molasses inoculum as my positive control. I will then compare the ethanol yield from each arm. My hypothesis is that the cane sugar substrate, which contains the most sugar, will outperform the carrot pulp substrate, which has residual sugars, and that the carrot pulp substrate will outperform the molasses inoculum with no substrate. I hypothesize that the carrot pulp substrate arm will validate the paper I am replicating (Khoshko, 2021) and will produce an average of 10 mL of ethanol. In all, should the experiment prove successful, it would further suggest the viability of pulp waste from the juicing industry as a potential industrial substrate for bioethanol production.

The concept of producing bioethanol from industrial food wastes is relatively new, with research labs only beginning to take interest a couple years ago (Barampouti et al., 2019). As stated above, methods, pretreatments, and fermenting organisms must be tailored to the specific composition of the substrate, thus highly efficient and refined procedures for the enormous variety of food waste substrates have yet to be developed (Roukas & Kotzekidou, 2022). However, a second generation process called simultaneous saccharification and fermentation (SSF), where enzymatic hydrolysis is performed during fermentation, has recently been shown to be generally effective among many different substrates (Chohan et al., 2020). A study by Aimaretti et al., 2012, details a procedure for the enzymatic hydrolysis and fermentation of carrot discards using separate hydrolysis and fermentation (SHF), generally found to be inferior to SSF for most substrates. Therefore, after the completion of my replicate study my next steps will be to update the procedure of Aimaretti et al. to instead utilize SSF, which I hypothesize will correspond to an increase in bioethanol yield from the fermentation process. 

Methods:

Substrate Preparation:

In the original study, carrot pulp waste was provided directly to the lab by industrial juice factories, however, as Berkeley Carroll had no access to substrate directly from a juice plant, carrot pulp was made from fresh carrots in the laboratory. Carrots were juiced with a Bagotte cold press juicer (link, Model: DB-001) to remove the sugar and nutrient content present within the juice.  The remaining pulp was thus a suitable replacement for juicing industry waste, with some possible differences being the type of carrot used and the mechanism of juicing. 

The pulp was collected and dehydrated for 24 hours at 100 ℃ in a Hamilton Beach multi-level food dehydrator (link, Model: 32100), which replaced a drying oven in the original procedure. As the purpose of this step is to dry the pulp into a more workable substrate, there are most likely no complications with this instrument substitution. 

The original experiment used an industrial grinder to turn the dried pulp into particles smaller than 200 microns in diameter, but to save on cost, a Secura fine grind coffee grinder (link, Model: SCG-903B) was used instead. After dehydration, the carrot pulp was passed through multiple rounds of grinding through the variable fineness coffee grinder (link, Model: SCG-903B) until a fine powder was achieved. 

The powder was then passed through an 80 mesh sieve (link), which has holes of 177 microns or 0.007 inches in size, with any powder left in the sieve being fed back into the coffee grinder for additional processing. Once collected, the sieved carrot powder (hereafter described as substrate) was stored in a Tupperware container until used in the experiments. 

Inoculum Preparation:

As instructed in the original experiment, molasses (128 grams, measured with an OHAUS CS2000 electronic scale (discontinued) and distilled water (371 mL, measured with a 150 mL graduated cylinder) were mixed in a 500 mL beaker such that an inoculum of 20 Brix was achieved. Sugarcane molasses (link) was supplemented for beet molasses as beet molasses is not commonly commercially available in the United States. The only major difference between the beet and sugar molasses, in so far as this experiment is concerned, is their sugar content, which is higher for sugar cane molasses than it is for beet molasses (LetCo, 2022). However, since the Brix (a measure of sugar content) of the inoculum is controlled, there should be no complications. The Brix of the solution was confirmed with a handheld Brix refractometer (link, USHC00020). The Brix refractometer had to be calibrated beforehand by placing three drops of distilled water on the lens and adjusting the tuning screw until the Brix level read 0. Afterwards, three drops of inoculum were placed on the lens of the refractometer to check the Brix, and more sugar molasses or water was added if the Brix was higher or lower than 20 Brix, respectively. The inoculum was then exposed to UV light in a UV sterilization hood (link, Model: 4777331) for 5 minutes to ensure no unwanted organisms would be present in the inoculum. Prior literature describes adding 1.5 grams of Saccharomyces cerevisiae over the course of 15 minutes, without any further explanation or instruction (Khoshkho et al., 2022). This was interpreted in the replicate experiment as adding 0.5 grams of Fleishmann’s ActiveDry S. cerevisiae (link) every five minutes over the course of 15 minutes while the beaker is stirred with the magnetic stir rod.

Experimental Setup:

Three 500 mL polypropylene bottles (link) were sterilized in a Tuttnauer tabletop autoclave (link, Model: EZ10P) at a steam temperature of 134 ℃ for 30 minutes, with one bottle each for the negative, positive, and experimental arms. The bottles, along with a 100 mL graduated cylinder, three 250 mL beakers containing 150 mL of distilled water each (measured with a 100 mL graduated cylinder), a weigh boat containing 10 grams of  substrate, another weigh boat containing 10 grams of cane sugar, and 3 different colored latex balloons were placed into a UV-sterilization hood and exposed to UV light for 5 minutes. The outside of the beaker containing the inoculum was then sterilized with Clorox wipes and introduced to the sterile UV hood. Using Clorox wipe sterilized long gloves (link) to reach inside the arm-holes of the hood, 150 mL of distilled water (from the premeasured beakers) and 50 mL of inoculum (measured with the 100 mL graduated cylinder) were added to each bottle. Afterwards, 10 grams of sugar was added to the positive control arm, 10 grams of substrate was added to the experimental bottle, and nothing was added to the negative control bottle. The positive control condition was not present in the original paper, but was added to ensure the experimental methods were sound.  Since S. cerevisiae produces ethanol from glucose, adding glucose in the form of cane sugar would certainly increase the ethanol yield over the negative control with nothing added, otherwise there would be an issue with the overall design of the experiment (Chang et al., 2018). For example, if the positive control yields significantly more ethanol, and the experimental arm generates more ethanol than the negative control, that ethanol would have to come from the 10 grams of carrot pulp. However, as the 10 grams of carrot pulp should always contain less sugar than the 10 grams of pure sugar, the experimental group should not outcompete the positive control. Conversely, while it is possible that the experimental group does not significantly outperform the negative control, it should never be outperformed by the negative control. If either are true, then there is an issue with preserving yeast viability, i.e. its ability to produce ethanol, across trials and/or experimental replicates. The bottles were incubated for 72 hours at 28 ℃ in a Quincy Labs analog-control incubator (link, Model: 12-140E). After incubation, the bottles were then subjected to a 80 ℃ hot water bath (link, Model: 1660504EDU) for 5 minutes to neutralize the yeast before being refrigerated at 5 ℃. Thus, the only variable between the positive control, negative control, and experimental arm is the type and amount of substrate from which the ethanol was produced, with all other variables held constant. 

Distillation:

Personal protection equipment, including lab safety goggles, a lab coat, and disposable gloves, were used while operating the distillation setup. As ethanol is a hazardous chemical for skin and eye contact, as well as potentially harmful if ingested, if any exposed part of the body came into contact with ethanol it was thoroughly washed with soap and water afterwards. As ethanol and its vapors are also flammable, no open flame or spark was let near the distillation setup while an active experiment was in progress. Storage in a separate flammables cabinet was unnecessary as the ethanol was stored in an ~95% water solution.

The original paper gave no protocol for the distillation of their inoculum after incubation, so a simple distillation procedure was adapted from the LibreText online chemistry textbook (Nichols, 2022). The procedure consisted of using a Chemglass organic chemistry lab set kit (link, CG-9800-02), Electromantle heating mantle (link, CMU050/EX1), and Flinn Scientific ring stands (link, Model: AP4550) and clamps (link, Model: AP1034) to distill the contents of each of the bottles for about 30 minutes at 91 ℃. While handling and adjusting the heating mantle, heat resistant lab gloves were worn. This would ensure that almost all of the ethanol, along with a significant portion of water, would be extracted from the culture as ethanol has a boiling point at 77 ℃, and while water has a boiling point of 100 ℃, water and ethanol create an azeotrope when mixed together in solution, and thus sometimes boil off together. A V-Resourcing handheld ethanol refractometer (link, Model: B07KLPFNBR) was then used to find the ethanol to water ratio of the distillate as done in Rijal, 2020 and Pornpunyapat et al., 2014. Calibration and measuring steps for this refractometer were identical for the Brix refractometer as previously mentioned above. Samples were stored within 50 mL Falcon tubes with a layer of Parafilm under the caps to prevent evaporation.

Data Analysis:

As the distillation removed all of the ethanol from the yeast fermentation, the total volume of the distillate was divided by the ethanol percentage of the distillate to calculate the total ethanol produced. This value can be averaged with the ethanol distilled from the other replicates of that arm.  This average can then be directly compared to the average ethanol produced from the other arms using a two tailed independent t-test (Megawati et al., 2022).

Section I Procedural Modifications:

A 2012 article by Shimokawa et al. shows that T. reesei enzymes are capable of producing significant amounts of fermentable sugars at 40 ℃ in SSF along with a thermotolerant yeast. The ActiveDry S. cerevisiae strain used cannot produce ethanol at this temperature for extended periods of time, therefore a new strain is used instead: Brewmaster Voss Kveik Ale Yeast strain (link) of S. cerevisiae. The strain provides thermotolerance at 40 ℃, which is a temperature that the T. reesi cellulase enzymes have been shown to operate at (if inefficiently) while also, despite being genetically distinct, is still a S. cerevisiae species. This allows for a more reliable direct comparison with the ActiveDry strain used in the replicate study (Amazon, 2021) (Foster et al., 2022) (Preiss et al., 2018). The preparation of the carrot substrate remains identical, as does the preparation of the 20 Brix molasses and distilled water mixture. As previously mentioned, 1.5 grams of the Kviek yeast is added to the 20 Brix (128 grams molasses, 371 mL distilled water) molasses inoculum over 15 minutes, with 0.5 grams added every 5 minutes while being stirred with a magnetic stir bar (link, Model #: AP1090).

Section II Procedural Modifications:

The negative control, positive control, and experimental arms of the original replicate study were prepared as previously mentioned except the 50 mL of ActiveDry inoculum in each bottle was replaced with 50 mL of the Kviek yeast inoculum.  In addition, a second  experimental arm was added where 0.1 grams (measured with an Ohaus precision balance (link, Model #: OB2148) of Carolina T. reesei cellulase powder (link, Product #: 853630) is introduced. This amount was chosen to match the 0.05% v/v dosage of T. reesei cellulase used in the Aimaretti et al., 2012 article. Thus, the only variable between the positive control, negative control, experimental arm, and cellulase added experimental arm is the no carrot substrate (negative control), 10 g of carrot substrate (experimental arm), 10 g of sugar (positive control), or 10 g of carrot substrate with 0.1 g of cellulase powder (cellulase experimental arm), with all other variables held constant. Incubation temperature was increased to 40 ℃ for all bottles for 72 hours, and the heat inactivation, distillation, and data analysis procedures remain identical to the original replicate procedure. 

Results and Discussion:

First (Failed) Replicate Procedure:

In the first procedure, the temperature was to be held at 77 °C by adjusting the heat of the heating mantle and the distance between the mantle and distillation flask as the temperature fluctuated; as ethanol has a boiling point of 77 °C and water has a boiling point of 100 °C, if performed correctly a negligible amount of water would enter the distillate.  By measuring the volume of the distillate, which should be almost pure ethanol, the amount of ethanol produced by a biological replicate could be determined. However, it was discovered during the distillation process that holding the temperature at 77 °C manually was extremely difficult, if not impossible, over the 30 minute distillation period, which led to the confounding variable of temperature fluctuation. As can be seen in Figure 1a, this temperature fluctuation led to periods when the temperature was high enough that during some distillations excess water boiled off and entered the distillate, and periods when the boiling intensity was too low for the ethanol to boil off. Furthermore, as the experiment had no mechanism by which to measure the concentration of ethanol within the distillate, the amount of water introduced into the distillate could not be determined, thus the volume of water in the distillate was another confounding variable. Thus, the data gathered in this experiment was influenced by too many confounding variables for clear analysis, which led to the development of a new, more precise procedure. 

Improved Replicate Procedure:

Based on the experience gained in the prior experiment, a new procedure was created using some modifications. Since the maximum temperature of the heating mantle is 91 °C, by keeping the heating mantle on full power during the entire distillation, the temperature was maintained at 91 °C without fluctuation, as can be seen in the temperature section of Figure 2a. As ethanol and water create an azeotrope when in solution, they tend to boil off together (Caravetta et. al., 2022). Therefore, a significant amount of water is introduced to the distillate during the course of distillation. To counter this, an ethanol refractometer was used to calculate the percentage of ethanol in the distillate, which, when divided by the total volume of the distillate, yields the volume of ethanol in the ethanol-water solution. In this way, the confounding variables of temperature fluctuation and water volume of the distillate were effectively removed from the experiment. In the two-tailed, independent t-test comparing the average volume of ethanol distilled from the negative control and the average volume of ethanol distilled from the experimental group, a t-score of 1.043 was calculated with 4 degrees of freedom.  At a significance level of p=0.05, a t-score greater than 2.776 or less than -2.776 is necessary for the relationship between the variables to be statistically significant. As such, the difference between the ethanol produced by the negative control and experimental group is not statistically significant. Furthermore in the two-tailed, independent t-test comparing the difference of ethanol production between the positive control and experimental group, a t-score of 3.982 was acquired with 4 degrees of freedom, with a t-score greater than 2.776 or less than -2.776 is necessary for the relationship between the variables to be statistically significant. As such, the difference in ethanol produced between the positive control and experimental group is statistically significant. In summary, the addition of carrot substrate did not significantly increase ethanol production, and the addition of an equal mass of sugar to the carrot substrate did significantly increase ethanol production as expected. These findings support the null hypothesis that ActiveDry yeast can not, on its own, utilize the carrot substrate for ethanol production.

Cellulase Study Procedure:

The cellulase study was a modification of the replicate experiment, using a different type of yeast (Kveik ale yeast), a higher incubation temperature, and adding another experimental group to be conducted in parallel as explained in the Procedure Modifications of the Methods Section. As such, both experimental groups were independently compared to the positive and negative control in two-tailed, independent, t tests like in the replicate study. For all t tests having four degrees of freedom, for an average difference in ethanol production to be considered statistically significant with a p value of 0.05, the t-score must be greater than 2.776 or less than -2.776. Compared to the negative control, the experimental group and cellulase experimental group had t-scores of 5.926 and 6.383 respectively, meaning that both experimental groups had a statistically significant increase in ethanol production compared to the negative control. Compared to the positive control, the experimental group and cellulase experimental group had a t-score of 1.114 and 1.063 respectively, falling within the range of not statistically significant, meaning that the ethanol production for both experimental groups tracked closely with the positive control. Finally, an additional fifth t-test was conducted to determine if there was a significant difference between the ethanol production of the experimental group and of the cellulase experimental group. Comparing the average ethanol production of the experimental groups to each other yielded a t- score of 0.156, meaning that the difference in ethanol production was not statistically significant. Therefore, the data suggests that the Kveik ale yeast, with or without the assistance of simultaneous saccharification and fermentation, was able to utilize the carrot substrate to generate as much ethanol as pure sugar.

Discussion:

The Khoshko et al., 2022 paper described an increased ethanol yield when they introduced a carrot substrate to a beet molasses inoculum containing S. cerevisiae. Using a procedure intended to replicate that study, the addition of a carrot substrate to a sugarcane molasses inoculum with ActiveDry S. cerevisiae did not increase ethanol production. Thus, the findings of the replicate study run contrary to the findings of the Khoskho et al., 2022 paper. Although more data should be collected before any definitive conclusion is reached, the findings of this experiment call into question the conclusions drawn by these authors. For the cellulase study, the data suggests that the Kveik ale yeast was able to utilize the carrot substrate to generate a similar amount of ethanol as in the positive control, which used an equal mass of cane sugar. Given the efficiency of sugar as a substrate for ethanol production, this result seems quite unlikely (de Almeida, 2023). Additionally, one of the hypotheses for this experiment was that the cellulase experimental group should outperform the experimental group in ethanol production. However, the data shows that there was no significant difference between the ethanol production of the experimental group and cellulase experimental groups. This result is ultimately not so surprising, since the cellulase works most efficiently at a temperature of around 55 °C, while the incubation temperature for the cellulase study trials was performed at 40 °C. Therefore, future studies should include repeating the cellulase study at higher temperatures (like 45 °C and 50 °C) to observe how it affects the ethanol production of the Kveik ale yeast. 

Preliminarily, these results appear to rule out saccharification as an explanation for the increased ethanol production when compared to the negative control, as the increase was similar for both experimental groups, with and without cellulase. Another possible explanation is that the Kveik ale yeast is better at using the residual sugars in the carrot substrate than the ActiveDry yeast, thus allowing it to outperform the negative control. While this is certainly possible, in isolation it remains unsatisfying. According to the USDA FoodData Central database, 100 grams of carrots contain 4.7 grams of sugars; removing the mass of water (as the carrot substrate is completely dehydrated) and adjusting for a mass of 10 grams means that the substrate should contain 4.02 grams of combined sugars (2019). Assuming the Kveik ale yeast is 100% efficient at utilizing this source of sugar, which in of itself is extremely unlikely, that still accounts for less than half of the required sugar to match the ethanol production of the positive control. An even more unlikely explanation would be that the Kveik ale yeast itself has its own internal mechanism of enzymatic hydrolysis, such as cellulase or pectinase, and are able to break down the polysaccharides in the carrot substrate as a source of sugar. Multiple studies characterizing the genetic differences between Kveik ale yeast and other brewing yeasts (such as S. cerevisiae) have been conducted without any mention of such enzymatic pathways (Preiss et al., 2018) (Cubilos et al., 2019) (Krogerus et al., 2018). In fact, Kveik ale yeast has been characterized as struggling to break down simpler polysaccharides like trehalose, allowing it instead to accumulate (Foster et al., 2021). Repeating this study with more trials and gathering more data points would be crucial before coming to any confident conclusion about the effect of carrot substrate on ethanol production as it pertains to the Kveik ale yeast. Likely avenues of inquiry could include investigating the effects of higher temperatures of fermentation, different cellulases and forms of enzymatic hydrolysis, and comparison of simultaneous saccharification and fermentation to separate hydrolysis and fermentation as it pertains to maximizing ethanol production.

The direct negative impact of burning fossil fuels on climate change is no longer in doubt.   Given the steady recent climb in global temperature and the already devastating impact on more severe storms, droughts, floods and wildfires, the race to develop climate friendly renewable energy must be a top priority for humanity.  Clearly a range of energy sources will be necessary, and therefore research into maximizing the production of greener bioethanol will be important.  Shifting bioethanol production to fully take advantage of agricultural waste products could fulfill the promise of bioethanol as a more green, climate friendly technology.  Efforts in this area have been promising, but much important work still needs to be done to make next generation green bioethanol a reality.

Citations:

Aditiya, H. B., Mahlia, T. M. I., Chong, W.T., Nur, Hadi, Sebayang, A.H., Second generation bioethanol production: A critical review, Renewable and Sustainable Energy Reviews, Volume 66, 2016, Pages 631-653, ISSN 1364-0321, https://doi.org/10.1016/j.rser.2016.07.015

Aimaretti, N. R., Ybalo, C. V., Rojas, M. L., Plou, F. J., & Yori, J. C. (2012). Production of bioethanol from carrot discards. Bioresource technology, 123, 727-732.

Alvira, P., Tomás-Pejó, E., Ballesteros, M., & Negro, M. J. (2010). Pretreatment Technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A Review. Bioresource Technology, 101(13), 4851–4861. https://doi.org/10.1016/j.biortech.2009.11.093

Amazon. (2021). Voss Kveik Ale Yeast Pack. [Product page]. Amazon.com. https://www.amazon.com/Voss-Kveik-Ale-Yeast-Pack/dp/B0973LBZ13/ref=asc_df_B0973LBZ13/?tag=hyprod-20&linkCode=df0&hvadid=598381276275&hvpos=&hvnetw=g&hvrand=6697452234356801877&hvpone=&hvptwo=&hvqmt=&hvdev=c&hvdvcmdl=&hvlocint=&hvlocphy=9067609&hvtargid=pla-1657837368459&psc=1

Azhar, S. H. M., Abdulla, R., Jambo, S. A., Marbawi, H., Gansau, J. A., Faik, A. A. M., & Rodrigues, K. F. (2017). Yeasts in sustainable bioethanol production: A review. Biochemistry and biophysics reports, 10, 52-61.

Babcock, B. A., & Fabiosa, J. F. (2011). The impact of ethanol and ethanol subsidies on corn prices: revisiting history. CARD Policy Brief, 11, 1-10.

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