Reduced Chemical Use in the Winery: a study of nitrogen analysis and use
Gary Zucca
Associate Professor
National University
gzucca@nu.edu
Carol Patterson Zucca
Owner and Winemaker
Zucca Mountain Vineyards
Abstract
Reducing chemical use in winemaking is a goal of sustainable winemaking practices.
Nitrogen is used in the fermentation process to provide nutrients to the fermenting yeast.
Low levels of nitrogen can cause slow or stuck fermentation. Also yeast with insufficient
nitrogen nutrients take different metabolic pathways that result in sulfur compounds that
swampy, sewer-like odors in wine. Conversely, high nitrogen levels can also be
associated with off odors in wine.
Most small wineries lack the resources to do nitrogen analysis themselves, so they resort
to checking for off odors by smell and adding nitrogen nutrients based on their past
experience or a commercial lab’s nutrient recommendations. Neither of these methods is
satisfactory and generally results in overuse of nitrogen. Recent advances in nitrogen and
the availability of used lab equipment on the internet has made nitrogen analysis
techniques available to small wineries. This article discusses how a small winery can
reduce the nitrogen usage in the vineyard and during fermentation. The equipment
needed for the analysis was purchased used through e-bay thereby recycling unwanted
equipment and saving considerable cost. The general nitrogen analysis techniques are
described in terms that can be understood by people without a biochemical background.
The authors also review the costs to develop a nitrogen analysis capability in a small
winery.
Key Words: Nitrogen analysis
Language: English
Reduced Chemical Use in the Winery: a study of nitrogen analysis and use
Introduction
Reducing the use of chemicals is one of the foremost goals of maintaining sustainability
and responsibility in a winery. Without accurate, inexpensive tests to measure the
concentration of potential harmful chemicals, an excess of these chemicals can be used
that are toxic, hard to eliminate or create problems in the community waterways
producing unwanted plant blooms and fish kills.
We chose nitrogen as a measurement that could be easily conducted in small wineries.
Typically, nitrogen, in different forms, is added in excess to assure proper fermentation of
sugar to alcohol by natural or added yeast. With the accurate measurement of ammonium
and nitrogen in the crushed grapes, the amount necessary for proper fermentation can be
determined. The amount of nitrogen measured in the fruit can also give valuable
information to grape growers regarding nitrogen application requirements in the
vineyards
Low levels of nitrogen in wine fermentation can result in slow or stuck fermentation or
off odors in wine, but excessive high levels of nitrogen can be associated with off odors
(Dukes and Butzke, 1998). Off odors usually are due to hydrogen sulfide being present
and the unpleasant swampy, sewer-like smells showing up in the wine. When yeast do
not have enough nitrogen, they use other fermentation pathways that release sulfur
compounds.
Most small wineries generally lack the resources to do nitrogen analysis themselves, so
they either check for off odors in fermentation by smelling must, or add DAP
(diammonium phosphate) and other yeast nutrients based on their previous experience in
making wine from various vineyards or a commercial lab’s nutrient recommendations.
None of the above methods are particularly effective. Lab analysis is costly, and the time
required to get results can be received too late to be able to take definitive action; adding
nutrients based when off odors are detected by smelling can be too late in the
fermentation process to be effective. Adding nutrients based on general recommendations
can be costly and inaccurate, resulting in a stuck fermentation. Climate change from year
to year can result in very different levels of nitrogen from the same vineyard, and
accurate measurements can help the viticulturist and winemaker identify problems and
find solutions in the vineyard and winery.
Recent changes in nitrogen analysis and the availability used lab equipment on the
internet has made nitrogen analysis techniques available to small wineries. This article
discusses how a small winery can reduce the nitrogen usage in the vineyard and during
fermentation. The equipment needed for the analysis was purchased used through e-bay
thereby recycling unwanted equipment and saving considerable cost. The general
nitrogen analysis techniques are described in terms that can be understood by people
without a biochemical background. The authors also review the costs to develop a
nitrogen analysis capability in a small winery.
Background
Nitrogen in wine must is available for yeast nutrients in two forms, ammonia and amino
acids. The total of these two sources are the yeast assimilable nitrogen (YAN). Both these
sources must be analyzed by separate methods. Ammonia analysis can be done through
an enzymatic diagnostic kit or by use of an electrode that attaches to most PH meters.
There are various ways to analyze the contribution of nitrogen in must from amino acids,
most of which either require expensive and unstable reagents or expensive equipment.
The method we will discuss in the analysis of nitrogen by orthophthaldialdehyde (NOPA)
method. The NOPA method requires reagents that are reasonably priced, stable, safe to
work with, and requires only a spectrophotometer and centrifuge as the major pieces of
analytical equipment.
Equipment and Reagents Required for Nitrogen Analysis
Equipment
As stated above, nitrogen from ammonia is done either through an enzymatic diagnostic
kit or an ammonium probe that can be attached to a standard PH meter. The cost of both
of these methods is dependent on the number of tests. The initial cost of the enzymatic
diagnostic kit is lower than the ammonium probe, but has a limited shelf life (1 month)
and can only conduct 30 tests. The nitrogen probe has a larger initial cost, but can be used
repeatedly over 2 years.
The only major piece of equipment required is a spectrophotometer. A spectrophotometer
consists of two instruments: a spectrometer for producing light of any selected color
(wavelength, measured in nanometers), and a photometer for measuring the intensity of
light. Most spectrophotometers have a wide range of wavelength, but the only
wavelength needed for this analysis is 335nm. The instruments are arranged so that
liquid in a cuvette (a small acrylic container to contain the sample) is placed between the
spectrometer beam and the photometer. The amount of light passing through the tube is
measured by the photometer. The photometer delivers a voltage signal to a display
device, normally a galvanometer. The signal changes as the amount of light absorbed by
the liquid changes. If development of color is linked to the concentration of a substance
in solution, then that concentration can be measured by determining the extent of
absorption of light at the appropriate wavelength (Rice, 2007).
We found spectrophotometers readily available on eBay at a wide range of prices.
Caution is needed when buying equipment on eBay. From our experience many sellers
have inherited the equipment from relatives and have no idea what it does or whether it
works. Just be certain to look at the seller ratings.
A centrifuge is used to separate suspended particles from the wine sample. Although this
piece of equipment is convenient and can also be found on eBay at a very reasonable
price, the sample can also be prepared by cold settling (Davis & Butzke, 1998).
Micropipettes and tips are used to transfer small amounts of material (1-50 uL) with
greater accuracy than is possible with standard pipettes. Pipette tips are disposable tips
used with the micropipette. Since these devices need to be accurate and calibrated, we
recommend buying them either new or from a reliable source.
Table 1 below illustrates the equipment needed for the analysis, the shelf life of each, and
the sources available for purchase and the approximate cost range.
Table 1. NOPA Equipment Requirements
Equipment
Source
Ammonium diagnostic kit Vinquiry
Ammonium probe
Vinquiry
Centrifuge
eBay
Spectrophotometer
eBay
Micropipette
e-BayFisher
Pipette tips
Fisher
Cuvettes
Fisher
Cost Range
$135
$600
$40-$500
$35-$2,000
$50-$250
1000/$55
500/$87
Reagents
All reagents used in the analysis can be purchased from chemical labs (Fisher). Some
paperwork to insure the lab that you are a reliable company and will not use the
chemicals for illegal purposes is involved before you will be able to purchase the
reagents. Most of the reagents will have to be shipped as HAZMAT, which requires
additional cost. It will save money if you are able to pick them up at the lab.
Table 2. NOPA Reagent Requirements
Reagents
Source
Ethyl Alcohol (95%)
Fisher
Sodium Hydroxide 98% Fisher
Boric acid (99%)
Fisher
Isoleucine (98%)
Sigma
Aldrich
NAC (98%)
Sigma
Aldrich
Cost range
$120 / ltr.
$30 ea.
$55 / 500g.
$112 / 100g.
$115 / 1100g.
Overview of NOPA analysis method
The best way to summarize the NOPA analysis is that it is easier done than said. That is,
the explanation may seem a bit complicate but, once conducted, the analysis will become
straightforward. The basic analysis is as follows: The main reagent used for amino acid
nitrogen analysis, orthophthaldialdehyde, (OPA). OPA reacts with amino acids in a wine
sample to form a product whose light absorbance is proportional to the concentration of
amino acids in the sample. The absorbance in the sample is then compared with of the
absorbance a standard (isoleucine) that has comparable absorbance as the amino proteins.
The resulting comparison will determine the equivalent amount of nitrogen in the sample
in milligrams of nitrogen per liter of wine (mgN/L).
Let’s examine the NOPA procedure step by step. The first step is to make two NOPA
buffer reagents, one containing OPA and one without. Second, two samples of clarified
wine juice are taken. One sample is treated with the buffer containing OPA and the other
is a blank sample without OPA. The blank sample is necessary because other non-amino
compounds absorb light and have to be accounted for (Dukes and Butske, 1998). The
absorbencies of the samples are then read in a spectrophotometer at 335 nm, and the net
absorbance of the sample is calculated by subtracting the absorbance of the blank sample
from the absorbance of the OPA treated sample.
The third step in the procedure is to create a standard by which to compare the
absorbance to an equivalent absorbance in the standard. The amino acid, isoleucine, is
used for a standard. Isoleucine was selected as a standard because it is safe, inexpensive,
has a long shelf life and, most important, has a light absorbance that corresponds to the
absorbance of nitrogen in the amino acids in wine.
The isoleucine standard is created by making four dilutions of isoleucine and measuring
the light absorbance of each dilution. The dilutions are converted to milligrams of
nitrogen and the absorbance of the sample is compared to the nitrogen equivalent of
isoleucine at a given dilution.
Table 3. Isoleucine Standard
μL lle
0
5
mM water
25 20
Concentration lle
(mM)
0
2
Equivalent N
concentration (mg/L)
0 28
15
10
25
0
6
10
84 140
The dilutions of isoleucine are made as indicated in Table 3 above. The equivalent
concentration of nitrogen is calculated by multiplying the concentration of isoleucine in
millimoles (mM) by 14, the atomic weigh of nitrogen. The absorbencies of the various
concentrations of isoleucine are then read in the spectrometer and plotted. An example of
this plot is below in figure 1 below.
Figure 1. Standard Isoleucine Plot
Absorbance
Standard Isoleucine Plot
1.5
y = 0.0059x + 0.3122
1
0.5
0
0
50
100
150
Mg Nitrogen (mg/L)
The plot in Figure 1 can be described by the equation y = 0.0059x + 0.3122 where:
y = the absorbance of isoleucine, and x = the equivalent mg/L of nitrogen.
Now that a standard by which to measure the nitrogen is established, the wine sample can
now be compared to the standard to calculate the amino acid nitrogen in the sample. As
an example, assume that the absorbance of a blank sample was 0.271 and the sample with
OPA was 1.51. Subtracting the absorbance of the blank from the sample with OPA yields
a corrected absorbance of 1.239. Since the absorbance of 1.239 is the value of y, we can
calculate x. Solving for x the formula is: x = (y -0.3122) / 0.0059. Substituting 1.239 for
y we get a value of 157 mg/L of nitrogen in our sample.
This section provides the basis for understanding the NOPA analysis. The following
section will discuss the details of how the analysis is done in the lab.
Yeast Assimilable Amino Nitrogen Procedure
1. Reagents
a. NAC (N-acety-L-cysteine) buffer was made using 1000 ml volumetric
flask which contained 3.886 g Sodium Hydrohide (NaOH), 8.368 g ortho
boric acid, and 0.816 NAC. The flask was made to volume using
deionized (DI) water
b. OPA (o-Phthalaldehyde) solution was made by adding 0.671 g OPA
dissolved and made to 100 ml with 95 % ethanol. The solution was then
added to an different NAC buffer made as #a
c. Reagents will be stable for 3 weeks at 4oC
2. Standard Curve
a. Add 1.5 ml NAC buffer into 1.5 ml cuvettes. Add 10 mM Isoleucine (Ile)
standard and water as follows:
Test Tube
1
2
3
4
10 mM Ile
0
5 uL 15 uL 25 uL
DI Water
25 uL 20 uL 10 uL 0
b. Add 25 uL 5 % OPA solution to each cuvette
c. Read absorbance at 335 nanometers (nm) wave length
3. Procedure
a. Centrifuge each sample to remove particulate matter
b. Use 2 cuvettes for each sample. First cuvette will be juice measured at fill
strength. The second will be juice diluted in ½ with DI water. This
dilution is allows a reading even if the nitrogen levels are too high
c. Add 1.5 ml NAC buffer into each cuvette
d. Pipette 25 uL of the sample into the first test tube. Add 12.5 uL of the
sample into the second test tube along with 12.5 uL DI water. Carefully
record the amount of juice added to each test tube and the sample source.
e. Read and record the absorbance of each sample without the OPA solution.
This will be the blank measurement to determine what the absorbance of
the NAC buffer and sample is without the OPA reagent.
f. Add 25 uL 5 % OPA solution. Mix each cuvette using a plastic sheet and
incubate at room temperature for 10 minutes.
g. Read absorbance @ 335 nm and record.
h. Subtract the blank absorbance value from the sample absorbance value.
4. Calculations
a. In Excel, set up the standard curve in 2 columns
b. Place absorbance reading in one column
c. Place the amount of Nitrogen mg/L in the second column. Mg/L nitrogen
are calculated by setting up proportions since 10 mM Ile contains 140
mg/L with out dilution (tube 4), tube 3 contains 84 mg/l, tube 2 contains
28 mg/l, tube one contains 0 mg/L. Place these values next to the
corresponding absorbance reading.
d. Make a chart using the 2 columns with mg N as the independent variable
and absorbance as the dependent variable. Select Scatter Plot. Go up to
the menu and select chart, add trend line, linear regression, options, check
Display equation on chart and Display r2 value.
e. You will get information about the fit of the regression and the formula to
calculate the sample absorbance and convert it to mg N/L, either
separately or set up a formula in Excel
f. Y = mx+b is the formula for determining a regression based on the
Isoleucine standard.
i. Y = absorbance of standard converted into mg/L nitrogen
ii. m = slope of the regression
iii. b = Y intercept
iv. x = mg N equivalents
g. Convert the above formula to solve for x using the formula x = (y-b)/m.
5. Terms
a. ml = milliliter = cubic centimeter = .001 of a liter
b.
c.
d.
e.
f.
g.
uL = microliters = .000001 of a liter
mM = millimoles = .001 of a mole
Mole = mass atomic weight of a substance in grams
Cuvette = container to hold the sample for use in the spectrometer
mg/l = milligram/liter which is equivalent to parts per million (ppm)
nm = nanometer = unit of measurement of wavelength in a
spectrophotometer. 1nm = 1 x 10-9 meter.
Conclusions
The purpose of this paper was to discuss the effects of nitrogen in wine fermentation and
how measuring accurate amounts of nitrogen present in the grapes at harvest can help
decrease the amount of usage in both the winemaking procedures and in the vineyard.
Because of increased availability of laboratory equipment and advances in nitrogen
analysis techniques, this procedure is now available to small wineries. The equipment and
reagents necessary for nitrogen analysis can be purchased for less than $2,000. Nitrogen
analysis can make wineries and vineyards more environmentally responsible and
sustainable, since much of the nitrogen in waste and runoff can be better controlled.
Also, the reuse of used equipment can recycle materials and reduce costs.
References
Dukes, B..C., and C.E. Butzke. (1998). Rapid determination of primary amino acids in
grape juice usine an o-phthaldialdehyde/N-acetyl-L-cysteine spectrophotometric
assay.Am J. Eno. Vitic. 49: 125-134.
Rice University. What is a spectrometer? Visited November 3, 2008 at:
http://www.ruf.rice.edu/~bioslabs/methods/protein/spectrophotometer.html
Weeks, S.M. & Henschke, P.A. Yeast assimilable nitrogen. The Australian Wine
Research Institute’s Analytical Service. Oct. 2007.