A REVIEW OF INORGANIC AND ORGANIC NITROGEN CONSUMPTION BY 
SACCHAROMYCES CEREVISIAE DURING ALCOHOLIC FERMENTATION IN 
WINE 
 
 
 
 
 
 
 
A Thesis 
Presented to the Faculty of the Graduate School 
of Cornell University 
in Partial Fulfillment of the Requirements for the Degree of 
Master of Food Science 
 
 
 
 
 
by 
Riley Sanchez 
May 2021 
 ii  
 
 
 
 
 
 
 
 
 
 
 
 
© 2021 Riley Sanchez 
 
ABSTRACT 
 
Nitrogen is an essential nutrient for all microbial growth.  Yeast cells require nitrogen 
to grow and complete fermentation.  While both organic and inorganic nitrogen are 
present in grape must, it is common practice in the wine industry to add nitrogen to 
fermentations.  Available products include an inorganic nitrogen source, di-
ammonium phosphate (DAP), and an organic nitrogen source, amino acid mixtures.  
Although both inorganic and organic nitrogen additions to wine fermentations are 
common practice in the wine industry, it is not well known how the type of nitrogen 
added impacts the fermentation.  This review examines current research to determine 
what impact, if any, nitrogen source has on alcoholic fermentations in wine and 
highlights the differences between inorganic and organic sources of nitrogen in 
alcoholic fermentations in wine.  
 
 
BIOGRAPHICAL SKETCH 
 
Riley Sanchez was born in San Diego, California.  She attended the University of 
Oregon as an undergraduate, where she earned her Bachelor of Science in Biology and 
Chemistry.  Upon graduation, she took a harvest internship at a winery in Edna Valley, 
California.  This internship sparked her passion for winemaking, blending together her 
two academic passions perfectly.  She continued to work in the wine industry, working 
in several regions of California and in the Marlborough region of New Zealand.  In 
2020, Riley began her Master’s study in Food Science and Technology, specializing in 
Enology under the supervision of Dr. Patrick Gibney at Cornell University.  She is an 
executive member of the Graduate Wine Society and a researcher in the Gibney Lab, 
which focuses on wine microbiology.  The following research project reflects her 
scientific enthusiasm for wine microbiology and her desire to improve the wine 
industry’s understanding of the importance of microbiology in winemaking. 
ACKNOWLEDGMENTS 
 
I thank my advisor, Dr. Patrick Gibney, for his support and mentorship throughout my 
study at Cornell.  I am grateful for the time spent in his lab alongside a great group of 
smart, kind, supportive colleagues.   I thoroughly enjoyed every minute of lab. 
 
I thank other V&E faculty members who helped me along the way, including Dr. 
Gavin Sacks, Dwayne Bershaw, and Erin Atkins, all of whom took the time to answer 
any question I had. 
 
I thank my colleagues in the Cornell V&E Food Science community and other 
students: Rachel Allison, Glycine Jiang, Joanna Haines, Margaux Mora, and Matthew 
Sheehan for their friendship and support, which has made my time at Cornell 
wonderful and memorable. 
 
I thank Peter Wetherwax, Nathan Carlson, and Susan Tousi, all of whom have 
mentored me and supported me in achieving my goals.  I thank my family members, 
Jackie Starr, Dr. Anna Seydel, and Audrey Cowardin, who have also been 
instrumental in me furthering my education. 
 
Lastly, I must thank my mom and dad, Janelle and Steven Sanchez.  Your 
unconditional love and support shaped who I am today and has allowed me to achieve 
my goals.  Thank you for being my role models in work, life, and love.  I am lucky to 
have you both as parents. 
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TABLE OF CONTENTS 
 
BIOGRAPHICAL SKETCH………………………………………………………….iii 
ACKNOWLEDGEMENT…………………………………………………………….iv 
INTRODUCTION……………………………………………………………………..1 
Nitrogen.……………………………………………………………...………..1 
Nitrogen in Fermentation…………..…...………………………….………...2 
Nitrogen Catabolite Repression…………………………………....………...2 
Yeast Assimilable Nitrogen………………..………………………………....3 
NITROGEN SOURCE UTILIZATION IN FERMENTATION.………...……………5 
Amino Acids.…………………………………………………………………..5 
DAP…………..…………………………...…………………………………...5 
Nitrogen Source Impact on Fermentation Kinetics……………...………....5 
NITROGEN SOURCE IMPACT ON SENSORY…………………………………….7 
Higher Alcohol Formation……………….…………………………………..7 
Acetate Ester Formation……………………………………………………..7 
Nitrogen Source Impact on Wine Sensory Parameters…………………….7 
FUTURE DIRECTIONS…………………………………..…………………………..9 
REFERENCES………………………………………………………………………..10 
 
 
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INTRODUCTION 
 
Wine is an alcoholic beverage that is produced from grape juice.  Yeast cells consume 
sugars present in the juice and produce ethanol and carbon dioxide as byproducts.  
Additionally, yeast cells release volatile aroma compounds from non-volatile 
precursors produced by grapes, influencing the aromatic qualities of wine (Bell and 
Henschke 2005).  Saccharomyces cerevisiae is the most common yeast used during 
wine fermentations.  Grape juice is a complex matrix containing many compounds that 
can be metabolized by yeast.  During fermentation, yeast cells metabolize these 
compounds in the juice that we then perceive as positive aroma and sensory 
characteristics (Bell and Henschke 2005).  In addition to the S. cerevisiae cells that 
perform this activity, there are many other yeast and bacterial species present in the 
juice that can spoil wine.  All of these microbes need nitrogen as a nutrient source to 
grow. 
 
Nitrogen: 
Nitrogen is an essential nutrient for all microbial growth.  For yeast growth in wine 
fermentations, nitrogen is an essential component of the nitrogenous bases which 
comprise the information-containing code in nucleic acids of DNA and RNA, is 
present in all amino acids, and is necessary for yeast to synthesize these molecules 
(Bell and Henschke 2005).  This means that for a yeast cell to grow and divide, 
nitrogen must be available for consumption.  Grape juice or must typically starts with 
a low concentration of usable nitrogen for yeast growth.  Thus, it is common practice 
to add a nitrogen nutrient source at the onset of fermentation to the juice or must, and 
again at one-third’s consumption of available nitrogen. In addition to influencing the 
yeasts ability to grow, nitrogen availability impacts the production of enzymes and 
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nucleic acids, with lower nitrogen availability corresponding to slower fermentations 
(Salmon et al. 1996) and higher nitrogen availability leading to microbial instability 
(e.g. excess nitrogen levels can support the growth of spoilage microorganisms after 
alcoholic fermentation is completed).  Both slower fermentations caused by limited 
nitrogen and microbial instability caused by an overabundance of nitrogen can lead to 
off flavor and aroma formation in the final wine (Waterhouse et al. 2016).   Because 
of the impact nitrogen has on fermentation kinetics and sensory characteristics, it is 
common practice in the wine industry for winemakers to add nitrogen to their 
fermentations. 
 
Nitrogen in Fermentation: 
There are two main categories of nitrogen formulation routinely used in the wine 
industry to supplement a fermentation: organic nitrogen and inorganic nitrogen.  
Organic nitrogen is comprised of amino acids, proteins, and peptides (Santamaria et 
al. 2020).  GoFerm and yeast extracts are commonly used to supplement organic 
nitrogen into fermentation.  Using an organic nitrogen source to supplement 
fermentation allows amino acids to induce their own uptake within yeast cells 
(Ljungdahl et al. 2009).  Inorganic nitrogen is often supplied as ammonium ions 
(NH4+) and is in the form of di-ammonium phosphate (DAP).  While there can be a 
small amount of NH4+  present in grape juice, most inorganic nitrogen found in a 
fermentation is added using DAP.  The yeast cell membranes have general amino acid 
permeases (Gap1p) that actively select for specific nitrogen sources (Feldman et al. 
2010).  These permeases will have different affinities based on the concentration of 
the nitrogen source. 
 
Nitrogen Catabolite Repression: 
2 
 
Nitrogen catabolite repression (NCR) occurs when a present nitrogen source reduces 
transmembrane protein uptake of a differing source, increasing the metabolism for the 
present source.  Glutamate and glutamine are important in the nitrogen metabolic 
pathway because they allow for the synthesis of necessary amino acids that lead to the 
growth of yeast cells.  Both glutamate and glutamine cause strong nitrogen catabolite 
repression effects because they are major nitrogen donors to amino acid synthesis.  
Their structures enable them to be continuously recycled in amino acid synthesis.  
NCR is used to determine whether or not a nitrogen source is preferred by yeast in 
alcoholic fermentation.  Most of what is known about nitrogen sources and their NCR 
effects is based on lab strains of yeast.  There is not a good amount of information 
available on wild yeast strains and their NCR effects.  A nitrogen source’s ability to 
reduce the uptake of a different nitrogen source and increase the yeast cell’s affinity 
for its own uptake defines what a preferred source versus non-preferred source is 
during alcoholic fermentation.   
 
Yeast Assimilable Nitrogen: 
Yeast assimilable nitrogen (YAN) is the fraction of nitrogen that yeast can utilize for 
growth.  In winemaking, YAN refers to the fraction of nitrogen that yeast can utilize in 
alcoholic fermentation conditions.  In some industries, such as the brewing industry, 
the usable nitrogen by yeast is known as free amino nitrogen (FAN).  YAN is a 
combination of both organic and inorganic nitrogen sources.  For organic nitrogen 
sources, L-amino acids except for lysine, histidine, proline, and cysteine, can be 
utilized by wine yeasts in fermentation conditions for their nitrogen source 
(Waterhouse et al. 2016).  Lysine, histidine, proline and cysteine are considered yeast 
non-assimilable nitrogen because their higher molecular weight coupled with 
Saccharomyces cerevisiae lack of significant proteolytic activity in fermentation 
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conditions makes these amino acids unusable during fermentation (Bell and Henschke 
2005).  Saccharomyces cerevisiae, specifically, can utilize ammonium ions and free 
alpha amino acids (Bell and Henschke 2005).  While different yeasts have different 
YAN requirements, typically a juice or must is considered to have a low level of YAN 
if the concentration is <100 mg/L, a moderate level if the concentration is 150-350 
mg/L, and a high level if the concentration is >600 mg/L (Bisson 1991, Ough 1991, 
Henschke 1993, Jiranek 1995, Beltran 2004).  Yeast cells are immobile, forcing them 
to interact with the source of nitrogen that is closest to their cell membrane.  However, 
yeast can select different nitrogen sources preferentially through the use of permeases, 
which are protein transporters encoded by multiple different genes in the yeast 
genome.  General amino acid permeases, such as Gap1p, are used to bring amino acid 
nitrogen sources into the cell, while permeases such as Mep1p, Mep2p, and Mep3p are 
used to select for NH4+  (Waterhouse et al. 2016).   
 
  
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NITROGEN SOURCE IMPACT ON FERMETNATION KINETICS 
Amino Acids: 
Amino acids are an organic source of nitrogen that is present in must.  Some amino 
acids are preferentially utilized regardless of an inorganic source’s presence (Prior et 
al. 2019).  While some amino acids are preferentially selected, there are several amino 
acids that are not utilized at all during fermentation, such as proline.  If an abundance 
of amino acids are present in must, yeast will not turn on the pathway to synthesize 
their own amino acids, but rather will import the existing amino acids (Prior et al. 
2019).   
 
DAP: 
DAP is an inorganic nitrogen source containing two ammonium ions per molecule.  It 
is well documented that an addition of DAP at growth phase increases population size 
of yeast (Bely et al. 1990a).  Increased population size is often correlated to increased 
fermentation kinetics and decreased off-aroma production (Bely et al. 1990a).  In a 
study by Arias-Gil et al 2007, ammonium addition decreased consumption of aspartic 
acid, alanine, and arginine while addition of amino acids increased the consumption of 
aspartic acid, alanine, and arginine.  This study suggests that DAP may have NCR 
effects in yeast cells when present, displaying a preference by yeast for inorganic 
sources of nitrogen.  Lab strain experiments have supported the idea that ammonium is 
a preferred nitrogen source in fermentation conditions (Ljungdahl and Bertrand 2012).     
 
Nitrogen Source Impact on Fermentation Kinetics: 
Current research suggests that the nitrogen source alone does not impact fermentation 
kinetics, but rather the nitrogen concentration of the source has a large role in 
determining fermentation kinetics.  In research conducted by P. Seguinot et al. 2018, 
5 
 
nitrogen source did not impact fermentation kinetics, with inorganic and organic 
fermentations progressing at the same rate.  They found that the timing of nitrogen 
addition in combination with concentration of nitrogen added had the most impact on 
fermentation kinetics.  Similarly, a 2021 study by Santamaria et al.  revealed that there 
are no significant differences in fermentations based on type of nitrogen added.  
Instead, both studies point to the yeast strain’s specific nitrogen requirements as the 
determining factor for having high vigor fermentations.  In addition, a study by Roca-
Mesa et al. found that at the right concentration, source did not matter.  They also 
found that at too low of a concentration (under 120 mg N/L) or too high of 
concentration (in excess of double the required amount by the specific yeast strain) of 
nitrogen impacted fermentation kinetics negatively, resulting in undesirable 
fermentation speeds and undesirable characteristics in the wine.  Overall, fermentation 
kinetics are less dependent on the type of preferred source added and are more 
impacted by the concentration of that source.   
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NITROGEN SOURCE IMPACT ON WINE SENSORY PARAMETERS 
Higher Alcohol Formation: 
Higher alcohols are defined as alcohols that have more than two carbons.  In wine 
fermentations, amino acids are metabolized through the Elrich pathway and converted 
into their higher alcohol form (Hazelwood et al. 2008).  Higher alcohol formation is 
an energetically favorable pathway and can be attributed to fermentations where the 
sole source of nitrogen was amino acids.  Low concentration of amino acids creates an 
imbalance to the equilibrium where higher alcohol formation is favored.  Formation of 
higher alcohols is favored in anaerobic conditions, such as fermentation.  Higher 
alcohols form because the yeast cell is taking the amine group from the amino acids to 
use to make other amino acids.  In doing so, it leaves behind the carbon skeleton of the 
amino acid, which becomes a higher alcohol.  
 
Acetate Ester Formation: 
It is commonly accepted that the easiest way for a winemaker to increase or decrease 
acetate ester formation is through yeast selection or manipulating fermentation 
conditions.  For example, anaerobic conditions favor acetate ester formation, while 
aerobic conditions inhibit acetate formation.  However, some research indicates that in 
addition to yeast selection and fermentation conditions, nitrogen source can impact 
acetate ester formation.  Amino acid rich juice leads to increased Aft1p activity and 
decreased Atf2p activity (Seguinot et al. 2018).  Aft1p is responsible for acetate ester 
production to a larger extent than available substrates.  This increase in Atf1p 
expression, in conjunction with other fermentation factors, can increase the total 
acetate esters formed.   
 
Nitrogen Source Impact on Wine Sensory Parameters: 
7 
 
Higher alcohol formation occurs in fermentations where amino acids are the abundant 
nitrogen source available to yeast.  In some cases, higher alcohol formation can lead to 
positive sensory characteristics, such as higher alcohols transformation into acetate 
esters that give Sauvignon Blanc its key aroma characteristics.  However, higher 
alcohols are mostly known as fusel alcohols, with aroma descriptors such as solvent, 
fusel, and boiled potato.  Depending on the type of wine being made, nitrogen source 
can be used as a way to limit the negative aroma characteristics in the final wine.   
 
Hydrogen sulfide is a negative aroma characteristic that is produced when either an 
organic source or an inorganic source of nitrogen is at low concentrations.  Hydrogen 
sulfide production diminishes when nitrogen is in excess.  This is supported by Wang 
et al. 2003, who observed a decrease in hydrogen sulfide production in musts with 
increasing YAN concentrations up to 250 mg/L.  Thus, a combination of nitrogen in 
slight excess and a limited sugar supply reduces this aroma fault (Jinarek et al.1995).  
Acetate ester form when the proper concentration of amino acids is available.  Too 
low of a YAN concentration leads to a decrease in the production of acetate esters.  
Acetate esters are responsible for the fruity, floral, and tropical aromas found in many 
wines.  Acetate esters are desirable in young wines, as their concentration diminishes 
over time as they are converted into their higher alcohol conjugate.  Overall, moderate 
levels of nitrogen yield the most desirable aroma characteristics (Torrea et al. 2011). 
  
8 
 
FUTURE DIRECTION 
There is a lot of available research on nitrogen’s importance as a nutrient in yeast 
growth and metabolism, the impacts of nitrogen additions on fermentation kinetics, 
and the impacts of nitrogen in producing pleasant sensory characteristics.  However, 
there is a lack of information on the impacts different types of nitrogen sources have 
on a variety of juices in alcoholic fermentation.  We do not know whether the nitrogen 
source, organic or inorganic, is more important for determining fermentation kinetics 
or impacting final wine sensory parameters positively in different varietal 
fermentations.  There are several experiments that could provide more information on 
this.  For example, an experiment could be to measure both fermentation kinetics and 
wine sensory parameters of juice fermentations that have organic, inorganic, and 
mixed nitrogen sources.  This could be done for several common wine grapes, such as 
Chardonnay, Sauvignon Blanc, and Pinot Noir.  These results can then be compared to 
see if there is any change between the different juices.  Not only will this provide 
information on the different needs of juices/musts, this will also provide clear results 
as to whether or not the nitrogen source specifically impacts fermentation.  Overall, 
the importance of nitrogen to wine fermentations is clear and there is still more to 
learn about how to utilize nitrogen source to positively impact fermentations.   
  
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REFERENCES 
 
Arias-Gil M, Garde-Cerdan T, and Ancin-Azilicueta C. 2007. Influence of Addition of 
Ammonium and Different Amino Acid Concentrations on Nitrogen Metabolism 
in Spontaneous Must Fermentation. Food Chemistry 103: 1312-1318. 
 
Bell SJ, and Henschke P. 2005. Implications of Nitrogen Nutrition for Grapes, 
Fermentation and Wine. Australian Journal of Grape and Wine Research 11 (3): 
242–95.  
 
Bely M, Sablayrolles JM, and Barre P. Automatic detection of 
assimilable nitrogen deficiencies during alcoholic fermentation in enological 
conditions. J. Ferm. Bioeng. 70:246-252 (1990). 
 
Feldman H. 2010. Yeast: Molecular and Cell Biology. Wiley-VCH, Weinheim. 
 
Hazelwood LA, Daran JM, van Maris A, Pronk J, and Dickinson J. 2008. The Elrich 
Pathway for Fusel Alcohol Production: A Century of Research on 
Saccharomyces cerevisiae metabolism. Applied and Environmental 
Microbiology. 74 (8): 2259-2266. 
 
Henschke P, and Jiranek V. 1993. Yeast: Metabolism of Nitrogen Compounds. In: 
Wine Microbiology and Biotechnology. Research Gate, no. August: 77–164. 
 
Jiranek V, Langridge P, and Henschke P. 1995. Regulation of Hydrogen Sulfide 
Liberation in Wine-Producing Saccharomyces Cerevisiae Strains by Assimilable 
Nitrogen. Applied and Environmental Microbiology 61 (2): 461–67.  
 
Ljungdahl P, and Bertrand D. 2012. Regulation of Amino Acid, Nucleotide, and 
Phosphate Metabolism in Saccharomyces Cerevisiae. Genetics 190 (3): 885–929. 
 
Monteiro F, and Bisson L. 1991. Biological Assay of Nitrogen Content of Grape Juice 
and Prediction of Sluggish Fermentations. American Journal of Enology and 
Viticulture  42 (1): 47–57. 
 
Prior K, Bauer F, and Divol B. 2019. The Utilisation of Nitrogenous Compounds by 
Commercial Non-Saccharomyces Yeasts Associated with Wine. Food 
Microbiology 79 (July 2018): 75–84.  
 
Roca-mesa H, Sendra S, Mas A, Beltran G, and Torija M. 2020. Nitrogen Preferences 
during Alcoholic Fermentation of Different Non-Saccharomyces Yeasts of 
Oenological Interest. Microorganisms 8 (2).  
 
Salmon et al Ref 
 
Santamaría P, González-Arenzana L, Garijo P, Gutiérrez A, and López R. 2020. 
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Tempranillo Red Wine Quality. Fermentation 6 (3).  
 
Seguinot P, Rollero S, Sanchez I, Sablayrolles JM, Ortiz-Julien A, Carole Camarasa 
C, and Mouret J. 2018. Impact of the Timing and the Nature of Nitrogen 
Additions on the Production Kinetics of Fermentative Aromas by Saccharomyces 
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Cerevisiae during Winemaking Fermentation in Synthetic Media. Food 
Microbiology 76: 29–39.  
 
Torrea D, Varela C, Ugliano M, Ancin-Azpilicueta C, Francis L, and Henschke P. 
2011. Comparison of Inorganic and Organic Nitrogen Supplementation of Grape 
Juice - Effect on Volatile Composition and Aroma Profile of a Chardonnay Wine 
Fermented with Saccharomyces Cerevisiae Yeast. Food Chemistry 127 (3): 
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Chichester, West Sussex, UK. 214-222. 
 
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