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1. Microorganisms found in Ethanol Production:
Bacteria
Yeast

2. culture (agar) plates

3. BACTERIA (SINGULAR: BACTERIUM)
are
Single-celled organisms
which are
Prokaryotes
Yeast
are a fungi
which are
Eukaryotes

4. THE MOST COMMON BACTERIAL SHAPES

5. TYPICAL PROKARYOTIC CELL

6. Typical Eukaryotic Yeast Cell

7. Source of bacterial contamination
Bacteria comes in
 On the corn, especially bad corn!
 On the trucks
Bacteria are in the water
 Well water
 Cooling tower water
Bacteria are in the air
 Higher in humid environments
 Summer time they thrive in moist environments
Bacteria are on your person
 Skin, mouth

8. CERTAIN SPECIES OF BACTERIA
AND WILD YEAST STRAINS LIVE
FAVORABLY IN ETHANOL
FERMENTATION CONDITIONS.
THEY COMPETE WITH THE YEAST
AND UTILIZE THE GLUCOSE.
THIS LOWERS THE ETHANOL
YIELDS AND INCREASES
UNDESIRABLE ORGANIC ACIDS.

9. Stress factors for yeast
• Temperature
– 95˚F at start of fermentation good
– Should lower temperature as alcohol concentration rises
• Ethanol
• CIP
• Sulfite
• Sugar
• Acetic and/or lactic acid
• Sodium
• pH
– Yeast perform well in acidic environments pH 3-4
– Acidic environment good for bacterial control

10. FERMENTATION PATHWAYS

11. VINEGAR (ACETIC ACID) IS MADE FROM ETHANOL
BY THE ACETIC ACID BACTERIUM, ACETOBACTER
ACETI
SAUERKRAUT IS MADE BY LACTIC ACID BACTERIA
NATURALLY PRESENT ON CABBAGE
PICKLES ARE MADE ESSENTIALLY BY THE SAME
PROCESS FOR SAUERKRAUT WITH ORGANISMS:
LEUCONOSTOC AND PEDIOCOCCUS

12. Comparison of relative efficiencies of
different types of respiration

13. AEROBIC RESPIRATION:
C6H1206 + 6O2 → 6H2O + 6CO2 + 2880 kJ
or
sugar + oxygen → water + carbon dioxide + heat

14. ANAEROBIC RESPIRATION WITH
ETHANOL FORMATION
(ALCOHOL FERMENTATION):
C6H1206 → 2CH3CH2OH + 2CO2 + 210 kJ
or
sugar → ethanol + carbon dioxide + heat

15. ANAEROBIC RESPIRATION WITH
LACTIC ACID FORMATION
(FERMENTATION):
C6H1206 → 2CH3CH(OH)COOH + 150 kJ
or
sugar → lactic acid + heat

16. Bacterial contamination

Bacterial infections can cause large losses in profit

Based on ~1% lactic acid growth at 13 wt% ethanol and $2
gal/ethanol

For example a 50 MMGY ethanol plant infection causes loss
of
1% loss = $1,000,000 per year
4% loss = $4,000,000 per year


1 organic acid molecule = 1 lost ethanol molecule C2H5 OH
1 lactic acid molecule = 1 lost ethanol molecule
2CH3CH(OH)COOH = 2CH3CH2 OH+ 2CO2

6C + 12H + 6O = 6C + 12H + 6O


17. Bacterial growth is difficult to control because they grow and
live in similar environment as yeast do.
Therefore, the bacteria compete with the yeast for nutrients
and produce unwanted byproducts.

18. LACTIC ACID BACTERIA (LAB)
Gram positive bacteria are Lactobacillus, Weisella,
and Pediococcus species.
Gram negative bacteria are Acetobacter and
Gluconobacter species.
Less common LAB contaminants:
Luconostoc, Streptococcus, Aerococcus,
Camobacterium, Enterococcus, Oenococcus,
Teragenococcus, Vagococcus

19. LACTOBACILLUS

20. Lactic acid on Hplc
• Lactic acid indicates bacterial contamination
• Risk stuck fermentation
• Primary source is a (LAB) lactic acid bacteria

21. Pediococcus
• Gram positive cocci
• Organized in pairs and Tetrads
• All strains appear to have built-in resistance to
high levels of penicillin and virginiamycin
 One hypothesis is that Pediococcus is more likely
when corn has been stored on the ground

22. PEDIOCOCCUS

23. ACETOBACTER
The bacteria are Gram negative and Gram variable
 no endospores
 catalase positive
 oxidase negative
 Is capable of metabolizing ethanol (Hoyer’s media)
 Durham tubes grow obligate aerobes incapable of
fermentation.


24. ACETOBACTER

25. ACETIC ACID
– Acetic acid “background” should be near detection
limit of HPLC
– Should strive to be below 0.05%
– Primary source heterofermentative bacteria
– Also aerobic acetic acid fermenters

26. GLUCONOBACTER
Gram negative ovoid or rod shaped
 fermentation (acetic acid (acetaldhydes)/vinegar)
 non-motile or lophotrichous flagella
 Catalase positive
 obligately aerobic organisms
 optimal growth temperature is 25-30˚C, however,
no growth occurs at 37˚C. They prefer pH of 5.5 6.0.


27. Gluconobacter

28. Weissella

29. WEISSELLA
Lactobacillus “like”
 Gram positive short rod
 Some strains highly resistant to virginiamycin
 (acquired resistance??)
 All strains susceptible to 0.5ppm of penicillin


30. Leuconostoc

31. LEUCONOSTOC
•
Ovoid cocci often forming chains
•
Gram Positive
•
Facultatively anaerobic bacteria
•
Catalase-negative
•
LAB bacteria
•
Pickles and saurkraut

32. – pH can significantly decrease during an infection
– Ethanol production will decrease with infections.
The severity of the infection and the time the
infection is present will dictate how much ethanol
will be lost
– Sugar usage will decrease meaning increased
residual sugars present in the DDGS and Wet-cake
causing a decrease in quality

33. Common contamination Sources
• Heat exchangers
• Yeast Prop
• CO2 header
• Fermentation – metal cracks
• Dead legs
• Leaking valves
• Water/recycle
• Air
• Pipe work
• Product storage

34. CIP
(CLEANING IN PLACE)
EVERYTHING NEEDS TO BE
CLEANED
– FERMENTERS
– HEAT EXCHANGERS
– MASH LINES
– BEER/MASH INTERCHANGERS
– YEAST PROPAGATION SYSTEM

35. Contamination sources
for bacterial infections
– Inadequate CIP
– Dead legs
• General cleanliness throughout plant
especially in mash, yeast props, heat
exchangers, and fermentation areas
– Poor grain
• Bacteria present in low numbers on good
grain
• Bacteria present in extremely high numbers
on bad grain

36. NO PRACTICAL WAY TO CIP
CO2 HEADER
ENTIRE MASHING SYSTEM
METHANATOR BACTERIA FLOAT OUT TO COOK WATER
SYSTEM
WATER TREATMENT SYSTEM

37. BACTERIALCIDAL OR
BACTERIOSTATIC




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
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Antibiotics
can reduce or kill bacteria
Can be very specific
Commercially available
Can be expensive
Creates resistance
Antibiotic companies often
can help lab to help id
resistant strains






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Alternatives:
Hop Acids substitute for
antibiotics
Very expensive
Chemical washes
Steam, Bleach, Hydrogen
peroxide, Caustic,
Chlorine dioxide, Iodophor,
Ammonium biflouride
Lower in cost but not
selective
Destroys yeast cells

38. GRAM STAIN
1. Primary stain: crystal violet stains all
cells purple
2. Mordant: Gram’s iodine crystallizes
purple stain in cells
3. Decolorizer: 95% ethanol dissolves lipid
layers in cell walls, allows crystallized
purple stain to wash out
4. Counterstain: safranin enters vacant
cells turning them red

39. GRAM STAIN

41. GRAM STAIN PROCEDURE

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Innoculate organism onto the slide by placing a drop of DI
water on the slide using sterile loop.
Place slide on warmer low until dry. Do NOT over heat.
Cover slide with Crystal Violet for 1 minute.
Rinse with DI water.
Cover slide with Iodine for 1 minute.
Rinse with DI water.
Drizzle alcohol over slide at a slant for 10 seconds.
Rinse with DI water.
Cover slide with Safranin for 1 minute.
Rinse with DI water.
Dry slide on warmer or sitting at a slant to air dry.

42. SERIAL DILUTION AND
STANDARD PLATE COUNTS
 Standard
plate count: One method of
measuring bacterial growth
 Agar
plate: A petri dish containing a
nutrient medium solidified with agar
 Serial
dilutions are used to dilute the
original bacterial culture before you transfer
known volume of culture onto agar plate

44. CALCULATION OF THE NUMBER OF BACTERIA
PER MILLILITER OF CULTURE USING SERIAL
DILUTION
Pour plate:
made by
first adding
1.0ml of
diluted
culture to
9ml of agar
Spread
plate: made
by adding
0.1ml of
diluted
culture to
surface of
solid
medium

46. ANOTHER WAY TO MEASURE
BACTERIAL GROWTH
Petroff-Hausser counting chamber
Bacterial suspension is introduced onto
chamber with a calibrated pipette
Microorganisms are counted in specific
calibrated areas
Number per unit volume is calculated
using an appropriate formula

48. MOST PROBABLE NUMBER (MPN)
Method to estimate number of cells
Used when samples contain too few organisms
to give reliable measures of population size by
standard plate count
Series of progressively greater dilutions
Typical MPN test consists of five tubes of each
of three volumes (e.g. 10, 1, and 0.1ml)

49. ANY QUESTIONS??