www.sustainabletechsys.com

1. Ethanol Plant Testing
-samples
-instruments
-astm
-tests
-services available
-training

2. 
Testing includes:
Grain
 Mash
 Ethanol
 Feed
 Wastewater
 Methanator
 Ethanol
 Microbiology ?
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3. Typical Inbound Grain Tests
Include:
Moisture (test weight lb/bu), crude
protein, crude fat or oil, starch, damage
and foreign material, dry matter, crude
protein, crude
fiber, calcium, phosphorus, sulfur, and ash.

4. Maize is
composed of
approx. 70 %
starch, 8 %
protein and 4 %
fat/oil. The rest is
composed of
water, fibers, sug
ar and
various mineral
nutrients.

5. Typical test I have
done:
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Moisture NIR
Crude Protein NIR
Starch NIR
Crude Fiber NIR
Crude Fat/Oil NIR
Ash NIR
Calcium
Phosphorus
Sulfur
Aflatoxins? KIT
Antibiotic residue? Exp
Wet Feed/Dry Feed/Syrup/Grain

6. Grain/Feed Data

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Moisture (overnight)
Crude Protein (LECO)
Crude Fat
Ash
Crude Fiber
Acid Detergent Fiber
(ADF)
Neutral Detergent .
Fiber (NDF)
Net E nergies
(NEL, NEM, NEG )
Total Digestible
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Nutrients (TDN)
Digestible Energy
Metabolizable Energy
Sulfur
Calcium
Phosphorus
Potassium
Magnesium
Sodium
Iron
Manganese
Copper
Zinc
Wet Distillers Grain (WDGS)
Package

8. Environmental Labs need
to be state certified:
Midwest Labs
SDK Labs
Servi-tech
*Magellan
Local??

9. Color (L* Value)
Moisture
Crude Protein
Crude Fat
Ash
Crude Fiber
Acid Detergent Fiber
(ADF)
 Neutral Detergent .
 Fiber (ADF)
 Net E nergies
(NEL, NEM, NEG )
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Nutrients (TDN)
Digestible Energy
Metabolizable Energy
Sulfur
Calcium
Phosphorus
Potassium
Magnesium
Sodium
Iron
Manganese
Copper
Zinc
Typical Tests for DDG Package

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Moisture (Vacuum oven)
Crude Protein (Kjeldahl)
Acid Hydrolysis Fat
Ash
Crude Fiber
Acid Detergent Fiber
(ADF)
Neutral Detergent .
Fiber (NDF)
Net E nergies
(NEL, NEM, NEG )
Total Digestible
Nutrients (TDN)
Digestible Energy
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Metabolizable Energy
Sulfur
Calcium
Phosphorus
Potassium
Magnesium
Sodium
Iron
Manganese
Copper
Zinc
Syrup Typical Package

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Whole Corn –
Moisture, protein, oil, and
starch.
Ground Corn –
Moisture, protein, oil, and
starch.
Mash – Total Solids. We will
develop other HPLC test if you
so desire
Cooker/Yeast Prop –
Brix, DP4, DP3, glucose, maltose
, glycerol, acetic acid, lactic
acid, ethanol
Fermentation Samples –
(same as above).
Beer Well - (same as above).
Wet Cake –
Moisture, protein, oil/fat, crude
fiber, Acid Detergent Fiber
(ADF), Neutral Detergent Fiber
(NDF), starch, and ash.
Dried Distiller Grains –
Moisture, protein, oil/fat, crude
fiber, ADF, NDF, starch, and ash.
NIR or Send Out?

12. MASH TESTING
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Solids
Brix
HPLC
FAN
pH
HPLC
NIR (as hplc)
DE
Yeast Counts
Microbiology
?
ETHANOL TESTING
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Acidity
Moisture Karl Fisher
Apparent Proof
pHe
Chloride Levels
Sulfate Levels
Denaturant
Heptane
Methanol
Specific gravity
Gum
IC
Copper
C of A’s ?
?

13. ASTM REQUIREMENTS
FOR ETHANOL?
In House or Send Out?

14. Required ASTM (D4806)
Testing on Ethanol and Denatured Ethanol
ASTM D5580 Aromatics
ASTM D5580 Benzene
ASTM D6650 Olefins
ASTM 5453 Sulfur
ASTM 5191 Vapor Pressure
ASTM D4052 Specific
Gravity
 ASTM D4815 Higher
Alcohols
 ASTM D130 Copper
Corrosion
 ASTM D86 Distillation Curve
to include: initial boiling
point 10% recovery, 50%
recovery, 90% recovery and
end point.
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ASTM D5501 Ethanol &
Methanol by GC
ASTM D7319 Chloride & Sulfate
by IC
ASTM D6423 pHe
ASTM E1064 Water
ASTM D381 Gum
(washed/unwashed)
ASTM D1613 Acidity
ASTM D5453 Sulfur
ASTM E1688 Copper
ASTM D4052 Specific Gravity
Hydrometer Proof
VISUAL Appearance
Calculation Denaturant
Concentration

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ASTM D4806 for ethanol used for fuel Property
Limits
Units
Ethanol 92.1 min. % v/v
Methanol 0.5 max. (5,000 ppm) % v/v
Water
1.0 max. (10,000 ppm)
% v/v
Solvent-washed gum 5 max. (50 ppm) mg/100 mL
Chloride ion
40 max. (40 ppm) mg/L
Copper content 0.1 max. (0.1 ppm) mg/kg
Acidity as acetic acid 0.007 max. (70 ppm) % w/w
Appearance
Visibly free of suspended or
precipitated contaminants
Denaturant
Minimum 1.96% v/v and maximum
4.76% v/v natural gasoline, gasoline components or
unleaded gasoline.

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*Quarterly Denatured Ethanol Testing Requirements:
Proof
Apparent Proof
ASTM E1064
Water by Coulometric Karl Fischer
ASTM D5501
Ethanol Purity
ASTM D6423
Determination of pHe
ASTM D381
Existent Gum
ASTM D512
Chloride ion
ASTM D1613
Acidity of Ethanol
ASTM D1688
Copper
ASTM D5453
Sulfur
**Monthly Denatured Ethanol Testing Requirements:
Proof
Apparent Proof
ASTM E1064
Water by Coulometric Karl Fischer
ASTM D5501
Ethanol Purity
ASTM D6423
Determination of pHe
ASTM D1613
Acidity of Ethanol
Optional testing for an additional:
ASTM D5827
Sulfate by Ion Chromatography
***Denaturant Testing Requirements:
ASTM D5580
Benzene, Total Aromatics
ASTM D6550
Olefins
Optional testing for an additional::
ASTM D5453
Magellan E85 and Ethanol Testing

17. 
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ASTM D 4806
Standard Specification
for Denatured Fuel
Ethanol for Blending with
Gasolines for use as
Automotive SparkIgnition Engine Fuel
ASTM D 5798
Standard Specification
for Fuel Ethanol (Ed75Ed85) for Automotive
Spark-Ignition Engines
ASTM Standards for Fuel Ethanol
Performance Specifications

21. ASTM D4806 (California)
Parameter Limits Test Method Frequency
Each batch
“calculated”
Olefins, 0.5 D6550-00
vol%, max
Each batch
“calculated”
Aromatics, 1.7 D5580-00
vol%, max
Each batch
“calculated”
Benzene, 0.06 D5580-00
vol%, max
Quarterly
Third party
Sulfur, ppm 10 D5453-93
California Requirements Differ

22. 
California Denaturant Standards
Parameter Limits Test Method
Frequency
Olefins, 10 D6550-00 Each batch
vol%, max
 Aromatics, 35 D5580-00 Each batch
vol%, max
 Benzene, 1.1 D5580-00 Each batch
vol%, max
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23. Producer name and
location
 Traceable lot number
 Load date
 Quality results, limits
and test method
used
 Quarterly third party
lab results statement
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Certificate of Analysis

24. Shipping Samples:
DOT Information:
UN
1987, Alcohols, n.o.s.(e
thanol, gasoline),
Hazard Class 3, Packing
Group II
Shipping

25. DOT and Placards
UN1987;
Alcohols, (ethanol,
gasoline)
or
NA1987; Denatured
Alcohol
Fuel Ethanol E10 or E85?
Place card: 1987

27. DP
HPLC
DP1=glucose
DP2=maltose
DP3=maltotriose
DP4=maltotetraose
DP5=maltopentaose
DP6=maltohexaose
DP7=maltoheptaose
DP8=maltooctaose
DP9=maltononaose
DP10=maltodecaose
Degree of Polymerization by HPLC

28. DP = Degree of
Polymerization
number of dextrose
molecules linked together
examples:
DP1, DP2, DP3, DP4…DP10…

29. The extent of starch hydrolysis is expressed as Dextrose Equivalent
(DE). DE is the percentage of glucosyl bonds that have been
broken.
Dextrose is another word for D-glucose.
So, DE will be low (4 - 6) at the beginning of the process such as
the Slurry tank outlet.
Meaning: Mostly large molecules exist and not many molecules of
glucose.

30. DEXTROSE
EQUIVALENT (DE)
When the DE is 100, all of the bonds between glucose units in
the starch have been hydrolyzed. About 10% of the starch
bonds are broken by the end of Conversion and up to half of
the bonds are broken coming out of Saccharification.
If half of the starch bonds are hydrolyzed the average DP is 2
and the
DE = 50
example: 25% DP1 + 25% DP3 + 50% DP2 = 50 DE
If a fourth of the starch bonds are broken the average DP is 4
and the
DE = 25.
DE determines the average DP length of the liquefied mash.

31. Estimation of DE from
HPLC results
%DP1 * 100 + %DP2 * 50 + %DP3*33+
%DP4*10
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% Solids
= est. DE

32. Milling
Mixing
Gelatinization
Cooking
Conversion
Saccharification
Fermentation
Distillation

33. Milling: Grind size effects
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A large particle size can be more difficult to gelatinize
since enzyme cannot react with the substrate efficiently.
This can result in yield loss (up to 10%).
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A small particle size (< 200 mesh) can also cause
problems:
Mixing of floured grain in water may be more
difficult.
Small particles will also cause problems in
centrifugation, evaporation, and drying.
May have some positive effect on theoretical yield
due to enzymes’ ability to react with starch.
Typical Size : Mill grain to 100% thru 16 mesh

34. Slurry: Gelatinization
The mash is cooked at 170 – 185 degrees to
gelatinize most of the starch and break the viscosity
enough to be pumpable.
Alpha-amylase and water molecules together are
randomly breaking down (or hydrolyzing) the
amylopectin (branches) part of the starch structure.
The alpha amylase produces a mixture of short
sugar molecules (e.g. maltose, maltotriose), and
some dextrin molecules (longer molecules of sugar).
4 - 6 DE mash

35. Liquefaction
Alpha amylase is denatured in
Liquefaction primarily because of
heat.
The heat further breaks down
starch molecules for the second
addition of alpha amylase.
DE of 12 – 14

36. Purpose of Fermentation:
To produce ethanol through the anaerobic
conversion of glucose by yeast.
51% of the glucose is converted to
ethanol and 49% is converted to carbon
dioxide
Ethanol Level at end of
Fermentation is typically 12 15% v/v.