2022 Ocean Water Fermentation

Funded Aug 2022

New Tides Distillery
Name: Bradley Abramson
Email: Abramson.Bradley.W@gmail.com
Chief Science Officer at New Tides Distillery and Sr. Scientist at Noblis
PhD, Cell and Molecular Biology (2018, Michigan State University); BS biotechnology and bacterial
genetics (2008, Purdue University)
Distilling Experience:
A strong background in yeast culture, genetic engineering, and a working knowledge of distillation on
a 15-gallon column still.
Research Experience:
20 years: J. Craig Venter Institute (Postdoctoral Fellow, 2018-2021); The Salk Institute for Biological
Studies (Postdoctoral Fellow, 2020-2021); Noblis (Sr. Scientist, 2021-current).
Please provide how your background experience will provide the foundation for your
Dr. Bradley Abramson is an interdisciplinary scientist with published research in molecular
and cellular biology, genetic engineering, genomics, and bioinformatics. These multiple skillsets have
allowed Dr. Abramson to experiment with novel culture methods of yeast strains and the ability to
understand the genetics underpinning Yeast mating and breeding. As a postdoctoral researcher and
Sr. scientist, Dr. Abramson manages scientists and bioinformaticians from multiple disciplines in
meeting client goals in multi-million dollar research grants. Dr. Abramson also has experience with
GS-MS which will be utilized in determining the flavor profile of the distillate and analysis of potential
carry-over contaminants.
Dr. Abramson, with Dr. Charlotte Miller (Chief Marketing Officer) and James Burdick (CEO),
founded New Tides Distillery, LLC in 2021 in San Diego, CA based on the idea that climate change
is a fundamental problem leading to freshwater scarcity. This is supremely evident in San Diego
where water is transported from land-locked states to California and recently as an investment in the
western hemispheres’ largest desalinization plant. San Diego is a coastal city where saltwater is
abundant and should be utilized efficaciously. Instead of refining saltwater to freshwater to support
the growth of yeast in a mash, the yeast can be cultured in the presence of seawater (specific gravity
1.021), a carbon source, and anoxic conditions to produce ethanol suitable for human consumption
while still being delicious. This is evident with proof of concept work with Sean Hallman at Shadow
Ridge Spirits Company.
New Tides Distillery proposes to develop a suite of Saccharomyces cerevisiae (hereafter
yeast) for fermentation of sugars to ethanol in seawater, producing a palatable distillate for human
The genome of yeast has been sequenced and characterized for growth in saline conditions
but generally without a focus on ethanol production for human consumption (Aguilera et al. 2010;
Corte et al. 2006). The osmotic and salinity response helps an organism adapt to altered solute and

ionic concentrations of different environments, such as seawater compared to freshwater. The
osmotic and salinity response has been well characterized in yeast and many of the genes defined
(Melamed, Pnueli, and Arava 2008). In some cases, genetic changes acquired for salt tolerance
come with a trade-off to ethanolic fermentation yield (Attfield and Kletsas 2000). Directed evolution
and refinement of cultured conditions allow the yeast to develop genetic changes better suited for
salty conditions and New Tides Distillery has harnessed a proof of concept strain and has shown this
strain is capable of producing at least 12% abv in a mash consisting of seawater instead of
freshwater (Table 1). Following distillation, no salt remains in the distillate and no off-flavors were
detected however further refinement is required to broaden the flavor profile of the yeast and test its
adaptability to other sugar sources.
Genomics and sequencing capacity has rapidly increased over the past several decades
which has reduced costs substantially and helped researchers understand the genetic attributes
leading to observed phenotypes (Wang et al. 2021). The genome can shed light on the gene content
of an organism and help describe the mechanisms behind the phenotypes of the organism.
Third-generation sequencing capabilities, like Oxford Nanopore, can yield contiguous chromosome
resolved genomes that also help to define structural variation, the genomic sequence, and even
epigenetic markers like genome methylation where all have been shown to alter gene expression
(Ding et al. 2020; Naish et al. 2021). With advances in whole-genome sequencing, a single yeast
strain’s genome can be determined for roughly 200$ using Nanopore’s Minion sequencing
Yeast has a 12 Mbp genome size consisting of 16 chromosomes which usually exist in a
single copy (haploid) in clonally replicating cells. The genome holds the information needed to
replicate, ferment, and importantly mate(Gabaldón 2020). Yeast cells have the capacity to mate and
have two mating types termed alpha an a. These two mating types are controlled by a single
genomic region that can change depending on the pheromones detected and although the genetic
process is complex it is well characterized (Strathern et al. 1982). Clonally replicating haploid yeast
cells pass on copied genetic material to daughter cells but, importantly, mating yeast cells contain
the gene content of the two parents (Gabaldón 2020) generating diploid cells that can shuffle the
genetic content of the resultant offspring. By mating two yeast cells with differing genomic content a
researcher can create new yeast strains with the attributes of both parents (Gabaldón 2020). During
sexual mating and hybridization, recombination between genomic regions takes place and results in
shuffling between genome copies obtained from the parents.
To mate yeast, selection criteria are required that provide the researchers with a means of
easily growing only the strain of interest on a selective medium. Often a single gene mutation is
characterized or introduced that knocks out an essential yeast pathway. The pathway can often be
rescued by supplementing the limiting metabolite in the growth media. When two strains of yeast,
containing different deleted pathways are mated and recombination occurs some of the future
offspring will contain the genes required to rescue the pathway lose. For example, if one yeast
cannot produce histidine and the other cannot produce methionine then the researcher must
supplement the growth media with those essential growth chemicals. Once mating occurs there is a
potential to recombine the two genomes of the parent yeast producing daughter cells containing the
functional copies for each pathway, therefore, the daughter cells do not require supplementation and
can be selected for on double-dropout media.
The yeast genome confers all the genetic information for mating as well as general
metabolism and fermentation. Generally, yeast prefers simple sugars because they are readily taken
up by yeast cells and require the fewest enzymatic steps to metabolize. Complex sugars, such as

starch commonly found in corn, is a long polymer that requires glucoamylase to break the complex
chain of unusable glucose into usable glucose monomers. The glucoamylase enzyme has
well-characterized activity at high temperatures in freshwater solutions yet it remains unclear if
commercial glucoamylase shows similar activity in seawater conditions.
Volatile chemicals produced by the yeast during the fermentation process can be sensed by
the human olfactory system (Waymark and Hill 2021). Some chemicals are considered pleasing like
vanillins while others can be undesirable (Pryde et al. 2011; Lahne 2010). Through mating, humans
have domesticated yeast and bred and selected strains with ideal volatile profiles. This can often
come with trade-offs where other desirable phenotypes are mated out and these can include
phenotypes such as salt tolerance. These flavors have been well studied however further analysis
by sensory panels can provide direct consumer detection of appealing flavors and directly improved
flavor profiles.
Given Dr. Abramson’s background in genomics, we will also sequence the genome of the
salt-tolerant strain as well as mated strains produced throughout the project. This will help determine
the mating type, genetic differences with similar publicly available yeast genomes that are not
salt-tolerant, and potential flavor pathways after mating. Several experiments will be performed to
identify the production capacity, flavor profile, and genetic makeup of salt-tolerant yeast. Initial
experiments will focus on testing our existing yeast strain in 5-gallon batches with various sugar
quantities in the mash to determine yield. These test batches will be further distilled and analyzed to
determine at what point diminishing returns occur on yield vs unwanted flavor compounds. This
quality control analysis will also be helpful for the analysis of potential contaminating ocean particles
such as plastics or algal toxins that may exist.
Preliminary Evidence:
A novel yeast strain fermenting in the presence of seawater instead of freshwater has been
shown by New Tides Distillery to be capable of producing distillate up to 160 proof in a 5-gallon test
still with the help of Shadow Ridge Distillery. For every 5-gallon mash, 5 gallons of saltwater can
replace 5 gallons of fresh water for 100% conservation in the mash step of alcohol distillation. We
propose to characterize and mate our existing salt-tolerant yeast strain with additional distilling
strains that have alternative flavor profiles (i.e. whiskey, sake, vodka), test the upper limits of ethanol
production in existing and new strains, and test alternative sugar sources (i.e. corn, rice, refined
sugar) yield.
To date, two separate distillations have been performed in synthetic seawater and pacific
ocean water with the salt-tolerant yeast strain (Table 1). Initial small-scale (1 quart) experiments
were performed using synthetic ocean water by mixing ocean salts with tap water. This synthetic
saltwater was used to compare ethanol productivity between the salt-tolerant strain and a
commercially available whisky strain after 6 days. Specific gravity was used to calculate percent
alcohol by volume and showed a substantially higher yield by the salt-tolerant strain.

Table 1: Table of salt-tolerant yeast growth and fermentation efficiency calculations in sugar
mash. Two separate batches have been performed to completion with various yields. Initially, a
single mash was made, split into batches, and pitched with either the salt-tolerant yeast or
commercially available whisky yeast.
1. MONTHS 1-3: Seawater in future experiments will mean Pacific coast ocean water acquired from
Cardiff beach or similar beaches. We will first test the yield of ethanol production by specific gravity
prior to and post mashing, as well as post distillation by the percent ethanol distilled in desirable
fractions. The salt-tolerant strain will be tested in various sugar content, mashes and various sugar
sources in seawater (corn, rye, barley, agave, wheat, potatoes, grapes). Test the efficiency of
common enzymes in seawater mash (i.e. glucoamylase) by iodine starch test. Test the growth of
salt-tolerant yeast and several commercially available distilling strains on YPD drop-out media to
define auxotrophic nature for future mating selection (e.g. HIS3, LEU2, URA3, MET17, or LYS2).
Ideally, each strain would be auxotrophic for a different metabolite to ease downstream mating.
Risk: Yeast could potentially not have any viable dropout selection genes and these would
have to be introduced via bioengineering (“Addgene: Yeast Prototrophy Kit” n.d.). The final distilled
product would not be GMO as it does not contain any DNA after distillation but would require an
additional regulatory hurdle for production.
3. MONTH 4-5: Determine the ideal distilling strains for mating. These will be determined based on
the volatile profile on the nose prior to distilling while grown on YPD plates as well as the auxotrophic
nature of the strains for ease of mating. The olfactory profiles will be used as comparison profiles for
future mated strains.
4. MONTH 6-9: Develop 5 mated strains with existing distilling strains with the salt-tolerant strain.
Following the determination of the auxotrophic nature of the strains, the 5 most suitable distilling
strains will be mated (“Yeast Mating, Sporulation and Tetrad Dissection” n.d.). The mated strains will
be maintained on double drop-out media, with seawater, and ~10% ethanol thereby decreasing the
chances of mating out desirable phenotypes.
Risk: Loci related to flavor may be genetically linked to salt tolerance and recombination
through mating does not yield altered flavor profiles. This seems unlikely given that flavor and salt
tolerance are generally quantitative trait loci and are controlled by many genes spanning the entire

6. MONTHS 10-11: A comparison of seawater contaminants and distillate will be performed. The
general concern is about toxins and plastics that are commonly found in the ocean and how they will
carry over to the consumer in the final product. We hypothesize no carryover will occur but has been
brought up as a concern by multiple potential consumers. This includes testing distillate for domoic
acid, a common algal bloom toxin (Anna McGaraghan, Raphael Kudela, Kendra Negrey, Corinne
Gibble, n.d.; Maeno et al. 2018). LC-MS/MS will be performed on seawater as a negative control
and the distillate to determine potential pollution carryover during the distillation process. Other
methods, such as GC-MS, may be used to determine the esterified profiles and will be determined in
final distilled products with the help of experts such as Spirit of Hven or other analytical labs. Using
pyrolysis-GC/MS, one can determine the plastic content of a sample. The most abundant plastics in
the ocean will be tested in the final distillate to determine if any carryover has occurred focused on
the most common petroleum-based plastics reported to appear in the ocean: polystyrene (PS),
poly(methyl methacrylate) (PMMA), polyvinylchloride (PVC), polyethylene (PE), polyethylene
terephthalate (PET), and polypropylene (PP) (Ribeiro et al. 2020). Furthermore, comparisons of the
salt-tolerant yeast strain grown in synthetic refined salt water and in freshwater will be distilled and
will be used for comparison.
Finally, we will sequence the genome of the salt-tolerant strain and mated strains to
understand the genomics of salt tolerance, fermentation, and flavor. In potential partnership with Dr.
Todd Michael at the Salk Institute, we will use Oxford Nanopore sequencing to resolve the genome
of the previously mentioned strains to contiguous chromosome arm resolution using a Flongle or
MinION flowcell. DNA extraction of yeast and sequencing are routine and several companies provide
kits with robust protocols (Collins et al. 2021). Finally, after sequencing, genes or loci known to be
associated with volatile flavor production will be investigated first using a pan-genome comparison
tool PanOCT. (Eder et al. 2018; Sutton et al. 2021). The pangenome provides a means of
comparing newly sequenced genomes to the complete variation of that species.
7. MONTHS 12: A series of sensory assessors chosen by experts convening sensory panels will be
used to test the final distilled product of the most appropriate strains. The sensory panels will be
performed and sensory assessors chosen by trained experts following standard practices (Rogers
2016). Samples may include the distillate from the initial salt-tolerant strain and newly mated strains.
This will help identify the success of mating and provides a baseline for the flavor profile. The
sensory panels will provide final summarization of findings of the salt-tolerant strains produced
based on a hedonistic taste profile. Submission of all required strains, mash bills, and clarification of
saltwater as a specialized ingredient by the TBB. Data will potentially be used for a patent
application with the eventual goal of providing yeast to interested distilleries for their own spirit
production alongside New Tides distillery. New Tides will initially focus on using the original
salt-tolerant strain for the production of high-proof spirits to supplement the seltzer market during this
$6,575.00 – Total funds requested.
$2,475.00 – MATERIALS: 50lbs grains (flaked maize, rye malt, flaked rice, raw sugar) (~$95 ea),
consumables for yeast culture (plates, media; $2000)
$1,100.00 – ANALYSIS: Instrumental analysis, inclusive of gas, columns, and consumables
estimated at $220/sample for 5 samples

$1,500.00 – GENOMICS: The cost of sequencing one yeast genome is roughly 300$ and this cost
will be used to sequence 5 salt-tolerant mated strains. Mass Spec will be determined with partners
with available capabilities. For example, Spirits of Hven may provide GCMS profiles of New Tides
distilled spirits with comparisons to existing spirit profiles. Specialized biochemistry labs, such as Dr.
Joesph Noel at the Salk Institute, have Pyrolysis-GC capabilities that may be used for testing known
$1,500.00 – SENSORY TESTING: The cost of a sensory panel is estimated for two products though
New Tides may provide cost sharing for these tests as additional tests are warranted.
New Tides Distillery will provide labor, professional time, yeast, distillery equipment, seawater, and
energy costs free of charge.
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