cara's microbiology presentation
DESCRIPTION
www.sustainabletechsys.comTRANSCRIPT
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Microorganisms found in Ethanol Production:
Yeast
Bacteria
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culture (agar) plates
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BACTERIA (SINGULAR: BACTERIUM)areSingle-celled organismswhich areProkaryotes
Yeast are a fungi which areEukaryotes
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THE MOST COMMON BACTERIAL SHAPES
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TYPICAL PROKARYOTIC CELL
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Typical Eukaryotic Yeast Cell
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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
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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.
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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
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FERMENTATION PATHWAYS
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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
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Comparison of relative efficiencies of different types of respiration
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AEROBIC RESPIRATION:
C6H1206 + 6O2 → 6H2O + 6CO2 + 2880 kJ
or
sugar + oxygen → water + carbon dioxide + heat
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ANAEROBIC RESPIRATION WITH ETHANOL FORMATION (ALCOHOL FERMENTATION):
C6H1206 → 2CH3CH2OH + 2CO2 + 210 kJ
or
sugar → ethanol + carbon dioxide + heat
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ANAEROBIC RESPIRATION WITH LACTIC ACID FORMATION (FERMENTATION):
C6H1206 → 2CH3CH(OH)COOH + 150 kJ
or
sugar → lactic acid + heat
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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
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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.
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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
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LACTOBACILLUS
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Lactic acid on Hplc
• Lactic acid indicates bacterial contamination
• Risk stuck fermentation
• Primary source is a (LAB) lactic acid bacteria
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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
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PEDIOCOCCUS
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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.
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ACETOBACTER
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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
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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.
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Gluconobacter
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Weissella
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WEISSELLA Lactobacillus “like” Gram positive short rod Some strains highly resistant to virginiamycin (acquired resistance??) All strains susceptible to 0.5ppm of penicillin
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Leuconostoc
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LEUCONOSTOC
• Ovoid cocci often forming chains• Gram Positive• Facultatively anaerobic bacteria • Catalase-negative • LAB bacteria• Pickles and saurkraut
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– 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
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Common contamination Sources
• Heat exchangers• Yeast Prop• CO2 header• Fermentation – metal cracks• Dead legs• Leaking valves• Water/recycle• Air• Pipe work• Product storage
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CIP(CLEANING IN PLACE)EVERYTHING NEEDS TO BE CLEANED– FERMENTERS– HEAT EXCHANGERS– MASH LINES– BEER/MASH INTERCHANGERS– YEAST PROPAGATION SYSTEM
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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
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NO PRACTICAL WAY TO CIP
CO2 HEADERENTIRE MASHING SYSTEM
METHANATOR BACTERIA FLOAT OUT TO COOK WATER SYSTEMWATER TREATMENT SYSTEM
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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
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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
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GRAM STAIN
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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.
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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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CA
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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
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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
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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)
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ANY QUESTIONS??