Microorganisms found in Ethanol Production:

Yeast

Bacteria

culture (agar) plates

BACTERIA (SINGULAR: BACTERIUM)areSingle-celled organismswhich areProkaryotes

Yeast are a fungi which areEukaryotes

THE MOST COMMON BACTERIAL SHAPES

TYPICAL PROKARYOTIC CELL

Typical Eukaryotic Yeast Cell

Source of bacterial contaminationBacteria 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

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.

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

FERMENTATION PATHWAYS

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

Comparison of relative efficiencies of different types of respiration

AEROBIC RESPIRATION:

C6H1206 + 6O2 → 6H2O + 6CO2 + 2880 kJ

or

sugar + oxygen → water + carbon dioxide + heat

ANAEROBIC RESPIRATION WITH ETHANOL FORMATION (ALCOHOL FERMENTATION):

C6H1206 → 2CH3CH2OH + 2CO2 + 210 kJ

or

sugar  →  ethanol  + carbon dioxide + heat

ANAEROBIC RESPIRATION WITH LACTIC ACID FORMATION (FERMENTATION):

C6H1206 → 2CH3CH(OH)COOH + 150 kJ

or

sugar → lactic acid + heat

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

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.

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

LACTOBACILLUS

Lactic acid on Hplc

• Lactic acid indicates bacterial contamination

• Risk stuck fermentation

• Primary source is a (LAB) lactic acid bacteria

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

PEDIOCOCCUS

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.

ACETOBACTER

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

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.

Gluconobacter

Weissella

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

Leuconostoc

LEUCONOSTOC

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

– 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 increasedresidual sugars present in the DDGS and Wet-cakecausing a decrease in quality

Common contamination Sources

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

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

Contamination sourcesfor 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

NO PRACTICAL WAY TO CIP

CO2 HEADERENTIRE MASHING SYSTEM

METHANATOR BACTERIA FLOAT OUT TO COOK WATER SYSTEMWATER TREATMENT SYSTEM

BACTERIALCIDAL OR BACTERIOSTATIC 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

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

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

GRAM STAIN

GRAM STAIN PROCEDURE

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.

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

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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

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

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 dilutionsTypical MPN test consists of five tubes of each of three volumes (e.g. 10, 1, and 0.1ml)

ANY QUESTIONS??