Industrial Fermentations

Major Industrial Fermentation Products

Non-Food Applications
Antibiotics Vitamins Amino Acids Food Applications
Organic Acids Citric Acid Lactic Enzymes Rennet Polysaccharides Xanthan Gum Oils and Fatty Acids Colors Flavors

Organic acids
Used as food acidulants Most versatile ingredients in industry because they are:
Soluble and hydroscopic Buffers and chelators

Organic acids produced by fermentation and commonly used as food acidulants include:
Citric Lactic Gluconic Propionic

Synthesis of citric acid

270,00 tonnes worldwide/year=$1.4 billion Produced by several molds and bacteria from a variety of substrates
Either Aspergillus niger or several molds or yeasts Glucose or sucrose as substrate

Citric Acid Cycle

A.A Fatty Acids Glucose
Glycolysis: Acetyl Co-A Pyruvate Citrate Oxaloacetate Reduce degradative enzymes
Overproduction of citric acid in A. niger requires several pathways and pathway modifications:
High flux of metabolites through glycolysis, glucose transport Block of TCA cycle reactions that degrade citrate

Methods for Citric Acid Production

Submerged or surface fermentation process, batch fermentation Production media: Beet molasses or glucose syrup as raw materials Nitrogen added as ammonium nitrate or sulphate Metals are often removed from raw material as high levels of iron appear to inhibit citrate production Fermentation: Inhibition of formation of long hyphae (which would result in dramatic increase of viscosity of fermentation medium) Low pH (pH is controlled at 2.2 2.6 by addition of NH3), dissolved oxygen concentrations, and temperature control are important

Citric Acid: Downstream Processing

Three separation methods
Direct Crystallization
Most successful with highly refined raw materials

Precipitation as calcium citrate tetrahydrate

Dominant process Calcium hydroxide is added to filtrate

Liquid extraction

Suspended particles are removed under vacuum Precipitate is washed to remove impurities Solution is concentrated using evaporators Fed to a crystallizer, Crystals are centrifuged, dehydrated, and ground

Schematic: Citric Acid Fermentation

Food Enzymes
Uses:
Cheese Bread Malt beverages Clarification of fruit and vegetable juices Meat tenderizing

Extracellular
Excreted by the cells (except glucose isomerase and invertase)

Fewer than 50 species of bacteria and fungi produce enzymes for the food industry

Food Enzymes: Examples

Lipases
Flavour and texture of cheese

Lactase
Production of lactose-free milk

Pectinase
Clarification of wine and fruit juice

Invertase
Confectionery

Proteases
Improvement in flavor and texture of cheese (Rennet) -- Meat tenderizer -- Baking, etc.

Industrial Enzymes from Aspergillus species

Enzyme -alfaAmylase, glucoamyl ase Pectinase Lipase Protease Catalase Glucose oxidase Cellulase Phytase Tannase Uses Enhancement of characteristics of baked goods, production of HFCS, production of alcoholic beverages Juice clarification, increase of grape juice extraction in winemaking Cheese ripening, flavor development & modification of cheese Beer chill proofing, meat tenderizing, soybean protein modification Removal of hydrogen peroxide in irradiated foods, commercial cake baking, & in cheese and milk Removal of O2 in high-fat products, canned foods, and bottled or carbonated drinks, enzymatic production of gluconic acid, kits Beer brewing, fruit processing, improvement of food texture, Increase of availability of organic phosphorus in animal feeds Hydrolysis of flavonols in instant and fermented tea

Rennet
Protease
Functions in cheese coagulation of milk proteins to curds Obtained from suckling calves stomachs
Reduction in available calves stomachs/Increase in cheese production Rennin/Chymosin

> 60% of cheese makers use fermentation product Gist-brocades

Maxiren
Identical to its natural counterpart (= Cost)

Organism: Kluyveromyces lactis

Natural habitat is milk/milk products/Also for production of lactase Simple recovery

Schematic: Rennet production/Downstream Processing

Fermentation

Acid Step Autolyses prochymosin into active chymosin

Removal of K. lactis biomass by centrifugation or filtration

Finished Product: Chymosin

Production of Polysaccharides
Uses:
Modify flow characteristics of fluids Stabilize suspensions Flocculate particles Encapsulate materials Produce emulsions

Most abundant carbon compounds in the biosphere Bacteria and fungi produce polysaccharides in amounts in excess of 50% of cell dry weight

Xanthan Gum
Microbial exopolysaccaride, synthesized by Xanthomonas campestris Soluble in hot or cold water Insoluble in most organic solvents High solution viscosity at low concentrations Good stability when exposed to freeze/thaw Unique rheological properties make xanthan gum a effective thickener and stabilizer
Used in sauces and syrups, ice cream, fruit drinks, salad dressings (easy to pour when taken from fridge)

Microbial Production of Xanthan gum

Organism and inoculum production Media preparation
Inexpensive and complex media (tap water, glucose, sucrose, and starch; acid whey from cottage cheese manufacture) Carbon is primary and nitrogen as secondary limiting substrate

Fermentation
Submerged, aerobic Xantham gum as secondary metabolite High viscosity as a potential problem

Schematic: Xanthan Gum Fermentation

Production of Xanthan Gum Ctd..

Downstream processing
Goal: To obtain xanthan gum in solid form Purification: reduce levels of other solids, improve functional performance, odor, color etc. Deactivate undesirable enzymes Modify chemical properties

Oils and Fatty Acids

Produced by oleaginous organisms
Oleaginicity: ability to produce acetyl-CoA Primarily yeasts and molds Candida and Saccharomyces cerevisiae

Acetyl Co-A

Malonyl-CoA (2 carbons) Fatty acid synthase Palmitate (16:0)

Citrate Oxaloacetate

Oils and Fatty Acids

Cons:
Increased Cost Low yield (~40%)

Pros:
Benefit countries that cannot grow plants that produce oil Value added commodities: from waste material, PUFA

Oils and Fatty Acids

Substrates:
Hydrocarbons and alkanes (can predetermine chain length) Mixed carbon sources: fatty acids (lead to high lipid contents) Molasses, whey (glucose or sucrose)

Media:
High carbon Limiting nutrient (usually nitrogen)

Batch culture

Oils and Fatty Acids

Two stage continuous
1st phase: growth 2nd phase: fat accumulation

Downstream Processing
Breakdown of cell walls to recover oil (acid/alkali) Wet or dry extraction Refined: degumming, refining, bleaching, deodorization

Oils and Fatty Acids

Fatty Acids Produced by Fermentation Common Name Butyric Valeric Shorthand C4:0 C5:0 Occurrence Butter Butter Butter

Caproic C6:0 Polyunsaturated Fatty Acids

Polyunsaturated Fatty Acids

Omega 3 and Omega 6 series
Structural components of the cell membrane Overall capacity for humans to synthesize is low Heart, circulatory disorders, cancer

Use:
Infant formula
FAO/WHO recommended inclusion of supplements High quality docosahexaenoic acid (DHA) Thought to be essential for proper brain and vision development in infants

Produced by heterotrophic algae

Crypthecodinium cohnii Mortierella alpina

Production of Polyunsaturated Fatty Acids

Solid substrate fermentation Rice bran substitute
Glucose or glycerol as a carbon source Whey, bran, or sweet potato residue

+ 2.3-5% N2 Initial moisture 65-68% pH range 6-7

Colors derived by fermentation

Natural: Colors extracted from foods or other biological sources as opposed to artificial and nature-identical colorant Challenges:
Purification Poor processing stability Low tinctorial strength High cost

Colors derived by fermentation

Carotenes and xanthophylls:
Industrial scale production of beta-carotene by unicellular algae is carried out in the US, Australia, and Israel

Riboflavin: Overproducers found among yeasts and fungi (green yellow tint of whey) Other colors: indigo, anthocyanins

Flavors derived by fermentation

Flavors are natural if obtained from natural substrates 50-100 microbial flavors commercially available Vanillin:
Natural ($4,000/kg) Fermentation ($1,000/kg) Nature-identical ($12/kg)

Raspberry ketone:
<1 mg/kg in raspberry Natural ($10,000/kg) Fermentation (Price???)

Other flavors: cinnamic acid, jasmin, eugenol

Flavors derived by fermentation

Significant opportunities for pathways engineering Shikimic Acid Phenyl propanoids and design of fermentation Cinnamyl alcohol processes (precurser Lignin Polymer feeding etc.)
Smoked Flavors Vanillin Eugenol

Shikimic Acid-Derived Flavors

Patent Process
A U.S. patent for an invention is the grant of a property right to the inventor(s), issued by the U.S. Patent and Trademark Office. The right to exclude others from making, using, offering for sale, or selling" the invention in the United States or "importing" the invention into the United States. There are three kinds of patents: Plant Design Utility

Patent Process
To receive a U.S. patent: File a provisional patent prior to disclosure File a patent application with the U.S. Patent and Trademark Office It is an expensive process: >$10,000!

The determination of priority is based on the premise that the first person to make an invention that is new, useful, and non-obvious is entitled to a patent Filing a provisional patent gives priority when filing for a patent

Patent Process: Laboratory Notebooks

Used to document the conception of an idea, progress of experiments, observations and results Used to prove a company or university's right to obtain a U.S. patent for an invention Permanently bound pages consecutively numbered Entries recorded in same color ink The pages should be signed and dated There should be little to no white space

Patent: High viscosity xanthan and process for preparing same

What is the problem? How does this invention solve the problem? Why is this new, useful, non-obvious?