Nitrogen as a yeast nutrient in alcoholic fermentation

Wine is a result of using yeasts in an anaerobic (oxygen-free) environment to convert sugars from pressed grape juice into ethanol in the two-step process illustrated in the figure below. The first step -- glycolysis -- results in the 6-carbon glucose being split into two 3-carbon pyruvate molecules. In the next step --- alcoholic fermentation (AF) - four atoms of oxygen and two atoms of carbon leave the pyruvate, resulting in acetaldehyde, which is subsequently converted into ethanol.

Alcoholic fermentation (Source: http://alcoholicfermentation.net/)

According to Fugelsang (Overview of yeast selection and malolactic fermentation on aroma, flavor and phenols), the yeasts (i) extract compounds from the solids in the must/juice in order to form the "characteristic metabolites of fermentation (alcohols, esters, fatty acids, carbonyls, etc.) and (ii) cleave cysteine-containing precursors such that volatile thiols (aroma component of several varieties) can be released. The yeast that receives most of the credit -- and does most of the work -- in alcoholic fermentations is a species called Saccharomyces cerevisiae (SC) which is "specialized in metabolizing media with high sugar content and small quantities of nitrogenous compounds" (Suárez-Lepe and A. Marota, New trends in yeast selection for winemaking, Trends in Food Science and Technology 23 (2012), 39-50.). Yeasts require nutritive support to allow the performance of the above functions in the hostile environment (ethanol-rich, acidic) of the fermentation tank. It is the nitrogenous aspect of that support that is the focus of this blog post.

Proteins are used by the yeast as (i) enzymes for the glycolytic pathway (indicated above), (ii) permeases in the cell membrane responsible for the transportation of compounds into the cells, (iii) cellular constituents (Kennedy and Reid, Yeast nutrient management in winemaking, The Australian and New Zealand Grapegrower and Winemaker, 537, October 2008). These proteins are synthesized by the yeast and nitrogen (N) is a key component in that process. According to Kennedy and Reid, "Efficient protein synthesis is needed for efficient sugar transport and overall yeast metabolism."

According to Schwarcz and Schoeninger (Stable Isotope Analysis in Human Nutrition, Yearbook of Physical Anthropology 34, pp. 293-321), almost 100% of exchangeable nitrogen is found in the atmosphere or dissolved in the world's oceans and is transferred from these environments into the biological system through the processes illustrated in the figure below.  Grape vine plants receive their nitrogen through this terrestrial nitrogen cycle.

Source: http://tolweb.org/notes/?note_id=3920

The nitrogen content of grapes are affected by variety, rootstock, climatic conditions, soil composition, vineyard management practices, fertilization, irrigation, rot incidence, and grape maturity (Kennedy and Reid). The yeast cells extract yeast assimilable nitrogen (YAN) from the grape must in the form of ammonia (preferred source of nitrogen for yeast growth as most easily assimilated) and amino acids and these are stored in the cell walls for later use. This extraction and storage of YAN is front-loaded in the AF process.

Nitrogen is required throughout the fermentation process with larger amounts being utilized during the exponential growth phase of the yesats and small amounts during the stationary phase. In some cases the grape must does not provide adequate amounts of assimilable nitrogen and is supplemented by added nitrogen in the form of diammonium phosphate (DAP). Juice levels of < 25 mg/L ammonia or < 150 mg N/L (measured as YAN) is considered nitrogen-deficient (UCDavis). Insufficient nitrogen can result in sluggish/stuck fermentations or sulfide formation (sulfur-like off-odors, mercaptans, and sulfur-containing acetic esters; the less assimilable nitrogen in the must, the more hydrogen sulfide will be produced). Supplements are best added incrementally and proportional to yeast growth (UCDavis).

Excessive nitrogen in the must can lead to elevated levels of ethyl carbamate (a supposed carcinogen) or urea excretion. The levels of nitrogen required for a successful AF is dependent on (Kennedy and Reid):

• Initial must YAN
• Yeast strain
• Fermentation temperature
• Initial grape sugar
• Other factors.

Amino acids are the building blocks of proteins and, when brought into the yeast cell, can be incorporated as is, transformed into a different amino acid, or broken down as a source of nitrogen or sulfur. The amino acids taken up by the yeasts from the grape must is primarily stored in the vacuole to be used for protein synthesis during yeast growth. Once access to inorganic nitrogen becomes difficult, the yeast begins to break down the stored amino acids to provide nitrogen for protein synthesis. The most important amino acids taken up by the yeasts are shown in the table below.

Amino Acid	Characteristics
Proline	Not metabolized appreciably by yeasts under winemaking conditions One of the predominant amino acids along with Arginine and Glutamine Main amino acid from low-fertilization vineyards
Arginine	One of the predominant amino acids Breakdown results in formation of urea and ammonia (During wine storage, urea can react with ethanol to form ethyl carbamate, a carcinogen). Located mostly in grape skin so processing practices could influence content in juice Main amino acid from low-fertilization vineyards
Glutamine	One of the predominant amino acids Favored by yeasts because it can be broken down to glutamate and ammonia

The less-important amino acids taken up by the yeast cells are alanine, serine, and theronine.

©Wine -- Mise en abyme

Have the World's best wine regions benefited from a previously unrecognized source of nitrogen?

Recently published research on sedimentary bedrock (Morford, Houlton, and Dahlgren, Increased forest ecosystem carbon and nitrogen storage from nitrogen rich bedrock, Nature, 9/1/2011, pp. 78-81) leads me to posit that some of the major wine growing regions of the world have been the unknowing beneficiaries of a heretofore unheralded source of nitrogen and that this has benefited them in their production of high quality wines.  Before expounding further on this hypothesis, I will provide some background.

As shown in the table below, nitrogen is an essential element in the growth of grape vines and an imbalance in its levels can lead to problems in the vineyard and/or in the winery.

Nitrogen and grape vines (Compiled from Grande Passione -- Soil minerals vs wine quality)

According to Schwarcz and Schoeninger (Stable Isotope Analysis in Human Nutrition, Yearbook of Physical Anthropology 34, pp. 293-321), almost 100% of exchangeable nitrogen is found in the atmosphere or dissolved in the world's oceans and is transferred from these environments into the biological system through the processes illustrated in the figure below.  The commonly held view is that grape vine plants receive their nitrogen through the terrestrial nitrogen cycle but the Morford study calls this into question.

Source: http://tolweb.org/notes/?note_id=3920

Nitrogen, as is the case for all plant nutrients, is sourced from the soil by the plant.  According to Christopher Bargman (Geology and wine in South Africa, Geoscientist 15(4), April 2005), soil is the major influence on the growth of the vine plant as it provides: (i) a supply of water; (ii) anchorage in the ground; and (iii) a source of nutrition. The classic soil profile is shown below.

Source: http://www.westone.wa.gov.au

Now back to the Morford, Houlton, and Dahlgren study.  According to the authors, sedimentary rocks contain considerably more fixed nitrogen than all of the fixed nitrogen in the biosphere due to the capture of "... organic matter in marine and freshwater sediments, where it is incorporated into rock as organic N or as ammonium in silicate materials."  Greater amounts of this rock-based N is contained in sedimentary rock (consolidated rock deposited in layers) than is contained in metamorphic ( a rock formed from preexisting solid rocks by mineralogical, structural, and chemical changes, in response to extreme changes in temperature, pressure, and shearing stress -- Answers.com) and igneous rocks (formed when molten minerals cool from a liquid into a solid).  Even though it has been known to geologists that this fixed N is secreted in bedrock, "... it is generally believed that rock N is not sufficiently important to alter the terrestrial N cycle."  The results of this study has shown otherwise.

The authors studied the nitrogen content of soils and forest foliage in forests that were underlain by both sedimentary and igneous bedrock and found the following: (i) the nitrogen content in soils and foliage that grow above sedimentary bedrock is 50% higher than soils and foliage that grow over igneous bedrock; (ii) nitrogen isotope values for rock, soils, and plants are indistinguishable among each other when located above a nitrogen-rich sedimentary bedrock but that is not the case for the same elements overlaying an igneous bedrock; and (iii) "forest responses to geological N inputs are manifested as higher foliar biomass production ..."

The authors conclude that "Our results raise the possibility that rock weathering may be a significant source of N to terrestrial ecosystems..." underlain by terrestrial bedrock.

I compiled the following table of wine regions that may and may not have benefited from the phenomenon described by the authors.  The table shows that some of the world's foremost wine regions are underlain by sedimentary bedrock and may have benefited from that "siteing.".

Additional study will be required to determine the benefits and disadvantages associated with subterranean nitrogen, the implications, and what, if anything, should be done to maximize its effects and minimize its disadvantages.  The information provided herein could be of benefit in making decisions regarding vineyard locations or vineyard retention in the case of a vine-uprooting program.