Secondairy Use: Exploring Fermentation of Ice Cream Waste Using Saccharomyces

cerevisiae and Kluyveromyces lactis to Produce Ethanol

Abstract

During the ice cream production process, a large amount of dairy waste is
generated. Ice cream waste is rich in sucrose and lactose, fat, and protein. Although
specific yeast strains have been shown to convert lactose in sweet whey into ethanol,
fermentation of ice cream waste into ethanol has not been reported. The goal of this
research is to explore whether ice cream waste can be converted into bioethanol using the
yeast strains, Kluyveromyces lactis and Saccharomyces cerevisiae. To study ethanol yield
created through fermentation, chemical analysis will be conducted on multiple batches of
ice cream waste to determine variation in composition across different producers. The
samples will then be fermented in trials using K. lactis and S. cerevisiae in single, co-, and
sequential fermentation to determine whether one or both strains will be most effective in
producing ethanol. This proposed research is important as it is the first step to creating a
value-added product from ice cream waste.

Project Rationale

Ice cream is a massively produced food item, with over 1.38 billion gallons
produced in the United States in 2022.1 However, thousands of gallons of ice cream waste
are created during this process as a result of unsellable product due to errors (incorrect
storage, for example) or contamination, among other sources in the production system.2
This ice cream waste is then stored on trucks and deposited in landfills or fed to pigs,
resulting in environmental concerns, such as contributions to global warming, and
additional costs associated with disposal.

Addressing the issue of disposed ice cream waste may help mitigate some of these
negative effects. However, there is no major method currently in use to deal with ice cream
waste in a climate net-neutral or positive way. Fortunately, fermentation may be the
solution and it may also provide economic benefits to producers.

Previous research on the fermentation of whey using yeast has shown that there
may be ways to utilize the dairy waste and transform it into ethanol. 3 Ethanol is a major
component of many disinfectants and fragrance products. As a skin-safe solvent, it is
often used to create sanitation wipes for medical and personal hygiene use. It is also
widely used in the creation of perfumes, as it comprises 70-90% of a product’s volume as a
body-safe dilutant.4 These industries generate over $520 million and $60 billion annually in
their respective markets.5,6
 Ethanol has also become a valuable item within the fuel industry, as a partial
substitute for fossil fuels in gasoline. It is projected that the biofuel market will rise at a
CAGR of 11.3% from 2024 to 2030, a massive shift when considering that the market in
2023 was worth $99.53 billion.7

With a growing movement to address environmental concerns and an exponential

demand for ethanol, this proposed research explores microbial fermentation as a pathway
to convert ice cream waste into a value-added product. The low-cost creation of a
byproduct with an increasing value may incentivize companies to implement a more
carbon-neutral approach of disposing waste and advance sustainable dairy agriculture.

Background

During ice cream production, for every 1 million gallons produced, about 50,000
gallons are considered waste.2 The reason for such a significant amount stem from the
nature of mass-produced ice cream as its production generates leftovers or residue
throughout the process. This includes unsellable contaminated ice cream, products which
may have been improperly stored or made, leftover dairy mix that is flushed out between
changes in production lines, and overproduction, among other causes.

With over 1.3 billion gallons of ice cream produced per year, roughly 65 million
gallons of ice cream waste are disposed using methods that do not create environmental
or financial positives.1 One common way of disposal is depositing ice cream waste into
landfills much like household waste. Although this is straightforward, ice cream waste is
an organic material which releases methane gas when decomposed, contributing to high
greenhouse gas emissions which intensifies climate change.8 Another way ice cream
waste can be disposed of is by being sold as pig feed to farms. However, this does not
generate much profit and may be inconvenient for ice cream companies to use depending
on how much waste needs to be stored for long periods of time.

Currently, there is no major research project directly related to the fermentation of

ice cream waste as a potential means to reduce environmental impact or to create value-
added products. However, a previous study conducted on the by-products of cheese and
yogurt production, such as sweet and acid whey, has shown that certain strains of yeast
can be used to create ethanol through fermentation. 3 For example, the yeast
Kluyveromyces lactis can convert lactose present in sweet whey into ethanol.

Ice cream waste contains large amounts of lactose and glucose which K. lactis can
use. The added sugar content also means that there are large amounts of sucrose which
can be metabolized by another yeast strain Saccharomyces cerevisiae to produce
ethanol.9 As ice cream tends to have sucrose levels ranging from 9-28%, this would
theoretically mean that the amount of ethanol produced through ice cream fermentation
would be larger than previous studies’ findings with whey. 10

A current challenge is that ice cream is not a standardized product as producers

may use different formulations and production methods. The chemical composition of
ingredients may also be different depending on the quality of ingredients used. As such,
the sugar content may widely vary, and the yield of the ethanol may be less than desirable
for some ice cream waste. This could discourage manufacturers from implementing a
fermentation-based approach if it does not create a guaranteed economic benefit for their
company. To overcome this challenge, a first step would be to understand the chemical
composition of ice cream from different manufacturers.

Of note, ice cream is an emulsified product, comprised of fat, water, proteins, and
sugar. Fat is a major component as ice cream is often comprised of more than 10% milk fat
based on its standard of identity.11 As a result, the distribution of fat globules within the
other components may prove to be a challenge for the yeast to access sugar for
fermentation, reducing or preventing ethanol production.

Previous research has shown that ice cream waste can be separated into fat and
other components using heat and centrifugation.12 However, it is unreliable to count on
current production plants having the necessary equipment for this separation. As a result,
this proposed research will focus on direct inoculation of the yeast strains to the ice cream
waste with no fat separation and minimal processing. Fat separation could be explored if
outcomes from this proposed research show that the current waste is a poor substrate for
yeast fermentation.

Description of Proposed Research

Overall, the goal of this proposed research is to explore whether ice cream waste
can be converted into bioethanol using microbial fermentation. My central hypothesis is
that the yeast strains, Kluyveromyces lactis and Saccharomyces cerevisiae, when used
alone or in combination can convert sugars present in ice cream waste to produce
bioethanol.

To test my hypothesis, I propose the following guiding research questions:

1. How does chemical composition vary between ice cream waste obtained from
small, medium, and large ice cream production facilities?
2. Can K. lactis and S. cerevisiae convert sugars present in ice cream waste into
bioethanol?
Objective 1: Identify and compare the chemical composition of vanilla ice cream
waste from small, medium, and large producers using standardized analytical
methods.

Hypothesis for Objective 1: I hypothesize that there will be no significant difference in

chemical composition of vanilla ice cream waste obtained from small, medium, and large
producers.

Task 1.1: Identification of ice cream waste samples based on plant size.

To note any potential differences in chemical composition of ice cream waste, a

system of classification will be created for samples. For standardization in this proposed
research, all ice creams samples will be vanilla and will have a total fat content of at least
10g per serving (according to nutrition labels). Vanilla was chosen due to the lack of
inclusions (for example, nuts, chocolate, etc.) to standardize and simplify the fermentation
process. Products with a fat content of 10 g and above were chosen as the primary focus of
this research is to use regular ice cream which is more widely consumed than reduced fat
or low-fat products. For each collection time point, samples of ice cream waste will be
collected based on the size of the production plant, including small, medium, and large*.

*Categorization by specific production size ranges will be determined after email

correspondence with companies.

Task 1.2: Sample collection of ice cream waste

The assigned ice cream waste will be collected from producers identified in Task
1.1. To account for batch-to-batch variation and differences in production time, samples
will be collected to represent multiple batches (n=4-6) and production time (n=4-6).
Samples will be transported on ice and processed immediately or stored at –20C until
further processing.

Task 1.3: Proximate analyses of sugar, fat, protein, and total solids in ice cream waste.

HPLC using a Aminex HPC-87H column will be used to measure the sugar content
(sucrose, glucose, lactose) present in the ice cream waste. Total amount of fat will be
quantified using Soxhlet extraction and protein levels will be determined using Bradford
Assay method. Gravimetric analysis will be used to determine total solids of the ice cream
waste. All analysis methods will be conducted using established protocols. Data from
proximate analyses will be recorded and used to establish a baseline chemical
composition before starting the fermentation process.
 Created with BioRender.com

Figure 1. Proposed methodology used in identifying and chemically analyzing ice

cream waste samples as outlined in Objective 1

Objective 2: Compare ethanol levels (g/L) produced by Kluyveromyces lactis and

Saccharomyces cerevisiae in 3- v. 7-day fermentation of ice cream waste.

Hypothesis for Objective 2: (a) I hypothesize that Kluyveromyces lactis and

Saccharomyces cerevisiae will metabolize the sucrose, glucose, and lactose present in ice
cream waste and produce ethanol via fermentation. (b) I further hypothesize that that co-
fermentation of K. lactis and S. cerevisiae will produce the greatest amount of ethanol.

Task 2.1: Conduct single fermentation, co-fermentation, and sequential fermentation of

ice cream waste using K. lactis and S. cerevisiae

Following proximate analysis, each collected sample will be divided into six micro
fermentation groups. In the single fermentation trials, one group of ice cream waste
samples will be inoculated with only K. lactis, and the second group will be inoculated with
only S. cerevisiae. In co-fermentation, both strains of yeast will be introduced to the
samples using a 1:1 ratio of inoculum (same starting cell concentration) at the same time.
In sequential fermentation, both K. lactis and S. cerevisiae will be inoculated with the
samples, but one strain will be added 24 hrs. before the other. For this proposed research,
both sequences will be tested.

The K. lactis and S. cerevisiae used for this research will be prepared by streaking
the yeast strains onto YPDA for 24 hours. Once they develop into 1 mm colonies, each
colony will be added to 5 mL of YPD liquid media and incubated overnight at 30C until
OD600=2-3.

To prepare the ice cream waste, each sample will be thawed at 30 C until it has
become liquid (~1 h) prior to fermentation. Ice cream waste will then be portioned into the
six different fermentation categories as triplicates and inoculated with the yeast
subculture depending on treatment type. Microfermentations will be incubated at 30 C
without shaking for 72 hours (3 days). In a second round of tests, the inoculated samples
will be stored for 168 hours (7 days).

Task 2.2: Proximate analysis of sugar, fat, protein, and total solids in fermented ice cream
waste

Using the same methods from Task 1.3, proximate analysis of sugar, fat, protein,
and total solids present in ice cream waste after 3- and 7-day fermentation will be
conducted. The final levels of sucrose, glucose, and lactose after fermentation will be
focused on during this analysis to estimate the amount of possible ethanol produced from
these sugars.

Task 2.3 Measurement of ethanol yield and fermentation efficiency

Using HPLC, the amount of ethanol produced through single, co-fermentation, and
sequential fermentation will be measured. Fermentation efficiency will then be calculated
from percent product yield as a function of initial sugar present in ice cream waste.

Task 2.4. Determining optimal fermentation period using time course trials

After comparing ethanol yield between 3- and 7-day fermentation, a time course as
well as the effect of agitation can be performed to find the optimal fermentation period and
condition. This will be done to determine at what fermentation time and condition would
produce the most ethanol.
 Created with BioRender.com

Figure 2. Proposed base methodology for microfermentations as outlined in

Objective 2

Impact of Research

This proposed research is important as it will encourage further exploration into

sustainability within the ice cream industry. As ice cream produces massive amounts of
waste, which is often thrown away or used in livestock, there is a high financial and
environmental cost associated with current disposal methods. However, through
microbial fermentation, ice cream waste could be utilized to create a valuable product
with the fermented foods industry that is expected to grow. By giving ice cream companies
economic incentive to reuse their by-product through a relatively low-cost fermentation
process, producers, consumers, and the environment can benefit from this research.
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