Industrial Microbiology and Fermentation

Industrial Microbiology

• Uses metabolic reactions to produce metabolites for products
• Focuses on microbial product production and environmental roles like biomediation and biodegradation.
• Microbes are efficient at producing large arrays of compounds, but in small amounts needed for their growth.

Fermentation

• Breakdown and re-assembly of biochemicals by organisms under aerobic or anaerobic conditions
• Essential part of the glycolysis pathway, vital for cell regeneration
• Nutrients are oxidized by glycolysis, and released electrons reduce NAD+ to NADH

Fermentation Products

• Waste products produced in the absence of oxygen (O2)
• Needed to regenerate NAD+ in the absence of O2.
• Pyruvate, produced during glycolysis, is metabolized into ethanol and carbon dioxide in alcoholic fermentation:
  • C6H12O6 → 2C2H5OH + 2CO2 + 2ATP

Alcoholic Fermentation

• Done by specific yeasts used in making alcoholic beverages.
• Other types of fermentation:
  • Distillation of alcoholic beverages (like Pistarea drinks)
  • Fermentation of grapes for wine
  • Fermentation of malted grains for beer

Yeast

• Unicellular fungi that reproduce asexually by fission
• Do not undergo true sexual reproduction
• Common in nature, found on/inside plants (husks and flowers)
• Causing decay of ripe fruits due to sugar consumption
• Ethanol production by fermentative yeasts may have evolved to inhibit bacteria competing for resources in ripe fruits.

Alcohol Timeline

• Developed from fruit-based drinks to mead and beer (honey)

Yeast Usage

• Different yeasts have different capabilities, based on sugar complexity
• Yeast uses more monosaccharides than trisaccharides
• Brewing yeast can directly digest starch
• During beer brewing, starches in barley are broken down into simple sugars through a process called mashing.

Beer Flavor

• Combination of changes at the molecular level in raw materials:
  • Grain
  • Hops
  • Yeast
  • Malt
  • Brewing Stages

Beer Ranges

• Alcohol by Volume (ABV) ranges from 3.1% to 30.1%
• Beers are classified as Ales or Lagers based on fermentation temperature:
  • Ales: Fermented at higher temperatures
  • Lagers: Fermented at lower temperatures
  • Specific yeast strains are used for each type.

Ale Styles

• Characterized as:
  • Chocotastey
  • Amber
  • Complex
  • Pale Ales
  • Stout

Lager Styles

• Characterized as:
  • Crisp
  • Smooth
  • Popular
  • Pilsner
  • Marzen

Raw Materials

• Main ingredients: Water, grains (primarily malted barley), hops, and yeast
• Beers contain 3-8% (v/v) ethanol.

Water

• Essential for brewing
• Water makes up 90-95% of beer
• Added to malt during mashing for enzyme activity
• Mineral content in water is important during mashing
• pH impacts enzyme activity
• Regional water analysis influences beer flavor
• Water type varies depending on the beer being brewed.

Water Hardness

• Amount of dissolved calcium and magnesium in water
• Higher mineral content makes water hard
• Water softening reduces the concentration of calcium and magnesium
• Soft water typically has a higher sodium chloride (NaCl) concentration, resulting in less calcium.

Common Salts in Brewing:

• Calcium Sulfate (Gypsum): Used for a drier, clean finish, like in Pilsner Lagers. Increases hop bitterness, ideal for IPAs.
• Calcium Chloride: Contributes to a fuller body and sweeter finish.
• Magnesium Sulfate (Epsom Salt): Increases sulfate and magnesium content, essential for yeast metabolism. Promotes healthy yeast growth and fermentation.

Water Hardness and Beer Styles

• Hard water is optimal for dark beers and stouts. The dark roasted malt contributes to higher pH, leading to a lower reduction in alkalinity.
• Soft water is suitable for pale lagers with a prominent hop character. Brewing with hard water would result in a harsh and stringent taste.

Malts

• Malted grains used in brewing
• Includes barley, rice, sorghum, wheat, oats, malt, etc.
• Source of starch (sugar) = Maltose
• Contributes to flavor, sweetness, and color of the beer

Hops

• Added during the boiling process
• Belong to the Humulus lupulus family, specifically Cannabis sativa
• Hop addition influences different aspects of the beer:
  • Beginning of boil: Contributes bitterness
  • End of boil: Contributes flavor and aroma
  • Produces pungent and aromatic character
  • Contains tannins, aiding in protein coagulation
  • Contains d-resin and β-lean for flavor and mold prevention
  • Contains pectin, which contributes to foam

Adjuncts

• Additional ingredients besides the main raw materials
• Includes rice, maize (corn), oats, wheat, barley
• Also includes chocolate, nuts, spices, and extra sugars
• Unfermented sugars like lactose are sometimes added to milk stouts.

Yeast

• Two types are used in beer brewing:
  • Ale yeast (Top fermenting) - Saccharomyces cerevisiae
  • Lager yeast (Bottom fermenting) - Saccharomyces pastorianus
• Primary metabolites produced during fermentation: Ethanol, CO2, and glycerol.

Flavor

• Imparted by hops and secondary metabolites produced by yeast
• Secondary metabolites can be good or bad:
  • Bad: Aldehyde (green apple), Diacetyl (100 malt dix), DMS
  • Good: Clove, Fruity (ester flavor)

Clarifying Agents

• Include Irish moss, gelden, and polyclar.
• Used to precipitate proteins and hop solids during the brewing process
• Found in trace amounts in the finished beer.

Brewing Process

1. Malting and Milling

• Milling: Crushing or grinding malt to reduce size and release fermentable sugars
• Malting: Converting barley into malt used for brewing. Prepares fermentable mashes from cereal grains.

Air Process of Malting:

• The process of producing enzymes to break down starch into sugar
• Quian's starches are transformed by soaking and germination
• This process converts the adron into algae and cooks it, making it ready for fermentation.

Enzymes in Malting:

• α-amylase: Breaks down amylopectin (long) branches of starches, creating long sugars. Optimal temperature: 67°C - 73°C.
• β-amylase: Breaks α-1,4 glucosidic bonds, degrading long chains into simple sugars. Optimal temperature: 55°C - 66°C.

Malting Steps:

• Grains are steeped for 1-3 days
• Cooled in humid air for germination (3-4 days)
• Dried in a kiln for up to 17 days
• Rest period of 3 weeks allows the grains to mature into malt.

2. Mashing and Wort Preparation

• Mashing: Mixing milled malt with water and heating for 2-3 hours in a mash tun. Enzymes digest starches and liberate sugars.
• Most abundant sugars found in wort: Maltose, maltotriose, glucose, sucrose, isomaltose.
• Other sugars identified: Isomaltose.

Lauterning

• The process of separating the mash into wort and residual grain.
• Residual grain is sparged with warm water to extract remaining sugars.

Wort Flavor:

• Wort is flavored during the boiling process in a wort kettle.

Reasons for Wort Boiling:

• Extraction of hop flavor and aroma
• Coagulation of remaining protein and partial hydrolysis
• Deactivation of enzymes active during mashing
• Caramelization of sugars
• Intensification of maillard reactions (reducing sugars and amino acids)
• Sterilization and concentration of the wort.

3. Yeast Fermentation

• Wort is cooled and yeast is added
• Yeast cultures for inoculation are usually recovered from previous fermentation tanks using treatments like:
  • Sodium bisulfite
  • Tartaric acid
  • Ammonium per sulfate
• These treatments reduce pH and eliminate bacterial contamination.
• Fermentation lasts for approximately 14 days.
• During fermentation, yeast converts sugars into ethanol, CO2, and glycerol.
• Secondary metabolites and diacetyl are also produced.
• After 14 days, CO2 production ceases as the yeast cells become inactive.

Yeast Types in Beer Production

• Saccharomyces cerevisiae (Top fermenting yeast) - Ales
• Saccharomyces pastorianus (Bottom fermenting yeast) - Lagers.

4. Post Fermentation

• Beer is stored at 0°C for several weeks
• This process precipitates protein, yeast, resin, and other undesirable substances.
• Resulting in a clear beer
• Esters are also produced which contribute to flavor and aroma.

Post - Fermentation

• After aging, the beer is carbonated with CO2
• Then cooled, sanitized, and packaged into bottles.

SAB Process

• The process of producing beer at South African Breweries (SAB).

Wort Production:

• 1. Milling: Malt is mashed in tuns for 2 hours to activate enzymes.
• 2. Lauter Tuns: Malt grains are processed, and wort is held.
• 3. Wort Kettle: Wort is boiled, and hops and adjuncts are added.
• 4. Whirlpool Kettle: Wort is rapidly spun to settle hops at the bottom. It is held for 20-30 minutes for hop flavor.
• 5. Separation: The wort is pumped out for separation.

Fermentation:

• Wort is placed into large fermentation vessels with cooling jackets
• Fermentation lasts 12 days at 14°C (Black Label: 15.8°C)

Maturation:

• After fermentation, beer is transferred to a quiller.

Filtration:

• Kieselguhr candle filters (diatomaceous earth) are used to clarify beer.
• Removes any residual yeast and trub.

Bottling:

• Beer is transferred to a bright beer tank.
• Concentrated to the correct volume.
• Carbonated before bottling and capping.

Beer Analysis

Colour

• Measured using a spectrophotometer.
• Standard Reference Method (SRM) measures color intensity.
• SRM Formula: 12.7 x DX A430 (LoG)

International Bitterness Units (IBU)

• Bitterness originates from α-acids extracted from hops during the boil.
• IBU reflects the type of hops used and when they were added.
• IBU Formula: 184 x A75nm x 47.