12 Industrial fermentation - Shroomery

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Industrial fermentationIndustrial fermentationProduction with microbesProduction with microbes

Industrial microbiologyIndustrial microbiology

The use of microbes in the productionThe use of microbes in the production

Start from an ancient timeStart from an ancient time

Large scale production Large scale production ––

Early 20Early 20thth century century –– beverage industry, vinegar, beverage industry, vinegar,

baking yeast, citric and lactic acidbaking yeast, citric and lactic acid

Late 20Late 20thth century century –– development of development of

biotechnology from the antibiotic industrialbiotechnology from the antibiotic industrial

Production with microbesProduction with microbes

Fermentation industryFermentation industry

Ability of microbes to produce Ability of microbes to produce ‘‘usefuluseful’’ productproduct

Usually a naturally produce substances for Usually a naturally produce substances for growth, and maintenance.growth, and maintenance.

Metabolites Metabolites

Primary metabolite Primary metabolite –– produce during growthproduce during growth

Secondary metabolite Secondary metabolite –– produce after growth produce after growth stagestage

Substrate

Product

Cell

Time

Substrate

Product

Cell

Time

Primary metabolite

Secondary metabolite

Trophophase Idiophase

Production with microbesProduction with microbes

Interdisciplinary areaInterdisciplinary areaMicrobiologistsMicrobiologists……..of course..of course

Molecular biologistsMolecular biologists

Chemists/biochemistsChemists/biochemists

Engineers Engineers –– hardware, softwarehardware, software

Lawyers Lawyers –– patent and permission patent and permission

InvestorsInvestors

Management team!Management team!

ProductsProductsI. Foods and foods relatedI. Foods and foods related

-- Fermented meat, Cheeses and milk products, Fermented meat, Cheeses and milk products, mushrooms, bakermushrooms, baker’’s yeast, coffee, pickles, single s yeast, coffee, pickles, single cellcell--proteins, vinegar, amino acids, vitamins, proteins, vinegar, amino acids, vitamins, alcoholic beveragesalcoholic beverages

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ProductsProductsII. Organic acidsII. Organic acids

-- Citric acids, Itaconic acidCitric acids, Itaconic acid

III. Enzymes and microbial transformationIII. Enzymes and microbial transformation-- Commercial enzyme, sterol conversionCommercial enzyme, sterol conversion

IV. Engineered microbesIV. Engineered microbes-- Insulin, human growth factorInsulin, human growth factor

Production with microbesProduction with microbesSteps in Industrial fermentationSteps in Industrial fermentation

Isolation of microbes that produce your interested Isolation of microbes that produce your interested productproduct

Screen for the best producing strain: naturally or Screen for the best producing strain: naturally or mutation.mutation.

Optimize production condition (growth Optimize production condition (growth ––basically) basically) in labin lab

Scale up from lab scale (up to 10 L) to industrail Scale up from lab scale (up to 10 L) to industrail scale (>10,000 L) (raw material?)scale (>10,000 L) (raw material?)

Food and food related productsFood and food related products

Whole cells productsWhole cells productsIncluding Including –– mushrooms and Singlemushrooms and Single--cell protein cell protein (SCP)(SCP)

Mushrooms Mushrooms –– fruiting body of mold!fruiting body of mold!Highly consumed every year Highly consumed every year –– high nutrition.high nutrition.Grow on decaying organic matter in soil or wood.Grow on decaying organic matter in soil or wood.

Stalk and cap = compact myceliumStalk and cap = compact myceliumGills = site of reproductive sporesGills = site of reproductive spores

E.g. E.g. Agaricus bisporusAgaricus bisporus (botton mushroom), (botton mushroom), Lentinus edulusLentinus edulus (Shitake mushroom)(Shitake mushroom)

Agaricus bisporus

Shitake mushroom Oyster mushroom Straw mushroom


SingleSingle--cell protein cell protein –– yeast, and cyanobacteriayeast, and cyanobacteria

Alternative food sources Alternative food sources –– mostly used now as mostly used now as

supplementary diet e.g. yeast is a source for B supplementary diet e.g. yeast is a source for B

Vitamins.Vitamins.

Organisms: yeast, Organisms: yeast, Spirulina Spirulina

Growing biomass and freeze dryGrowing biomass and freeze dry

Raw material from other industrial, farmingRaw material from other industrial, farming

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Food and food related productsFood and food related productsBakerBaker’’s yeasts yeast

Same process as in single cell protein Same process as in single cell protein


Cheese productionCheese productionCan produce from any milk.Can produce from any milk.4 phases: coagulation, separation, shaping 4 phases: coagulation, separation, shaping and ripeningand ripening

Coagulation: rennin enzyme or acids precipitate Coagulation: rennin enzyme or acids precipitate caseincasein

From calvesFrom calves’’s stomachs, engineered bacterias stomachs, engineered bacteriaAcid production by lactic acid bacteriaAcid production by lactic acid bacteriaCurd entrap bacteria, fat globules and other Curd entrap bacteria, fat globules and other material.material.

Cheese productionCheese productionAdditives (salt and herbs) may be added after Additives (salt and herbs) may be added after seperation or shaping.seperation or shaping.

Additives and shape effect property of cheese Additives and shape effect property of cheese productproduct

Ripening Ripening –– add flavor and lookadd flavor and look

Natural or inoculation of microbes e.g. Natural or inoculation of microbes e.g. Penicillium Penicillium roquefortii (roquefortii (spores are usually added into blue spores are usually added into blue cheese), and cheese), and Penicillium camembertiPenicillium camemberti is added to the is added to the surface of a cheese known as Camenbertsurface of a cheese known as Camenbert

Blue cheese

Brie

Gorgonzola

Swiss

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Yogurt and other fermented milk productsYogurt and other fermented milk productsYogurt, kefir, buttermilk, sourcreamYogurt, kefir, buttermilk, sourcreamYogurt usually use mixture of 2 cultures:Yogurt usually use mixture of 2 cultures:

Streptococcus thermophilusStreptococcus thermophilus: ini. acid production: ini. acid productionLactobacillus bulgaricusLactobacillus bulgaricus

0.5 0.5 -- 1.0% lactic acid 1.0% lactic acid Fermented at 28Fermented at 28--32 32 °°C C Taste due to acetaldehyde, diacetyl, and acetoin Taste due to acetaldehyde, diacetyl, and acetoin Product need to contains live culturesProduct need to contains live cultures

A mixed lactic acid and alcoholic fermentation.

Streptococcus lactis, Lactobacillus bulgaricus, yeasts

Kefir

This milk product is used for its medicinal therapeutic value.

Lactobacillus acidophilus

Acidophilus milk

Made with skimmed or partly skimmed pasteurized milk.

Streptococcus sp. Leuconostoc sp.

Cultured buttermilk

Cream is inoculated and leave to develop acidity

Streptococcus sp. Leuconostoc sp.

Sour cream

DescriptionMicroorganismsFermented Product

Some Foods Produced from Fermented Milk.

Industrial production of yogurt

Kefir Buttermilk

Food and food related productsFood and food related productsAlcoholic beveragesAlcoholic beverages

Accidental discovered from ancient human Accidental discovered from ancient human Saccharomyces cerevisiaeSaccharomyces cerevisiaeFermentation of sugar in fruit, grains and otherFermentation of sugar in fruit, grains and otherIn case of other materials In case of other materials ––need to convert need to convert polysaccharide to fermentable sugarpolysaccharide to fermentable sugarRaw materials are regionalRaw materials are regional…….variety of products.variety of products

Wine Wine –– grapes, fruitsgrapes, fruitsBeer Beer –– cereal grain cereal grain –– barley, sorgum, wheatbarley, sorgum, wheatOther Other –– rice wine (rice), teguila (agave cactus), vodka rice wine (rice), teguila (agave cactus), vodka (corn), mead (honey).(corn), mead (honey).

Tequila

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Fermented meatFermented meatUse to preserve foodUse to preserve food

E.g. salami, summer sausage, cured ham E.g. salami, summer sausage, cured ham

Thailand Thailand –– naam (fermented pork), fermented naam (fermented pork), fermented sausage, fermented fish, fish saucesausage, fermented fish, fish sauce……etcetc

Main organism Main organism –– Pediococcus serevisiae, Pediococcus serevisiae, Lactobacillus plantarum, Lactobacillus plantarum, other lactic acid bacteriaother lactic acid bacteria

Production of lactic acid to change pH of food Production of lactic acid to change pH of food

Production of probiotics in some strainsProduction of probiotics in some strains

Food and food related productsFood and food related productsFermented fruits and vegetablesFermented fruits and vegetables

Found around the worldFound around the world

Hundreds if not thousand type of productsHundreds if not thousand type of products

With bacteria, yeast and moldWith bacteria, yeast and mold

Depends mainly on native microbes associate with Depends mainly on native microbes associate with the plants.the plants.

Most included mixed culture fermentationMost included mixed culture fermentation

Conditioning by human to favor growth of Conditioning by human to favor growth of beneficial microbes beneficial microbes -- saltingsalting

Rhizopus oligosporus, Rhizopus oryzae, Lactobacillus delbrueckii, Saccharomyces rouxii

Soy sauce

Leuconostoc mesenteroides, Saccharomyces marscianus, Flavobacterium spp., Fusarium spp.

Coffee

Lactobacillus mesenteroids, Lactobacillus plantarum

Sauerkraut

Pediococcus cerevisiae, Lactobacillus plantarum

Pickles

Leuconostoc mesenteroides, Candida sp.Cocoa products

OrganismsProducts

Food and food related productsFood and food related productsFlavoring agents: amino acids and nucleotidesFlavoring agents: amino acids and nucleotides

Monosodium glutamate, inosinic acid Monosodium glutamate, inosinic acid –– flavor flavor enhancersenhancersOriginally use seaweed, fishOriginally use seaweed, fishProduce by enzymatic hydrolysis of yeast RNA Produce by enzymatic hydrolysis of yeast RNA ((Candida utilis)Candida utilis)Now direct fermentation = monosodium glutamate Now direct fermentation = monosodium glutamate and 5and 5’’--IMP (inosine monophosphate)IMP (inosine monophosphate)Brevibacterium ammoniagenesBrevibacterium ammoniagenes produce inosine produce inosine and then chemically phosphorylatedand then chemically phosphorylated

Improve flavor of fruit juicesAspartateAlanine

Antioxidant in powdered milkL-tryptophaneL-histidine

Added to plant derived food to inprove nutritional value

L-lysine L-methionineL-threonineL-tryptophane

Low-caloric sweetenerL-aspartyl-l-phenylalanineMethyl ester (aspartame)

Flavor enhancerSodium glutamate

Infusion solutions for surgery patientsMixed amino acid

UtilizationAmino acids

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Acetate75Brevibacterium flavum

L-lysine

Glucose>100Corynebacterium glutamicum

L-glutamic

Sucrose55Escherichia coli K12

L-threonine

Glucose39CorynebacteriumL-lysine

C-sourceApprox. yield (g/L)

OrganismAmino acid

Corynebacterium glutamicumCorynebacterium glutamicum E. coliE. coli

NonNon--food productsfood products

Enzyme productionEnzyme productionProduce in large scale around the worldProduce in large scale around the worldMajor producer Major producer –– Novozyme (44%), Genencor Novozyme (44%), Genencor Intl, USA (18%)Intl, USA (18%)

Cheese productionMicrococcusLipases

Fruit juice clarificationAspergillusPectinase

Cheese manufactureAlcaligenes,Aspergillus,Candida

Rennin

Starch liquefaction, brewing, baking, feed, detergents

BacillusBacterial amylases

Fructose syrup productionAspergillusGlucoamylase

Antitumor agentEscherichia,Serratia

Asparaginase

SweetenersBacillus, StreptomycesGlucose isomerase

Semisynthetic penicillinsEscherichiaPenicillin acylase

DetergentsBacillus, StreptomycesBacterial proteases

UseOrganismsEnzyme

NonNon--food productfood product1. Vitamins1. Vitamins 2. Antibiotics2. Antibiotics

3. Alkaloids3. Alkaloids 4. Steroids4. Steroids

5. Non5. Non--Steroid Hormones/cell regulators (cytokines):Steroid Hormones/cell regulators (cytokines):

1. Epidermal growth factor 2. Proinsulin1. Epidermal growth factor 2. Proinsulin

3. Insulin 4. Human 3. Insulin 4. Human growth hormonegrowth hormone

5. Somatostatin 5. Somatostatin 6. Interferons6. Interferons

7. Platelet7. Platelet--derived growth factorderived growth factor

8. Fibroblast growth factor 9. Tumor Necrosis Fa8. Fibroblast growth factor 9. Tumor Necrosis Factorctor

10. Other cytokines are coming on line all the time10. Other cytokines are coming on line all the time

NonNon--food productfood product

6. Blood coagulating factor XIII6. Blood coagulating factor XIII

7. The restriction enzymes7. The restriction enzymes

8. Other enzymes (e.g. ligase, DNA polymerases 8. Other enzymes (e.g. ligase, DNA polymerases

etc.)etc.)

9. Solvents9. Solvents

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How do the microbes produce these How do the microbes produce these products?products?

Genetically modified microorganismsGenetically modified microorganisms

Clone genes that encode protein of interest into Clone genes that encode protein of interest into

vectors and express this genes in microbes used in vectors and express this genes in microbes used in

production.production.

Able to produce high amount of compounds than in Able to produce high amount of compounds than in

original organisms with ease of manipulation.original organisms with ease of manipulation.

Microbes able to utilize waste from other process as Microbes able to utilize waste from other process as

a raw material for fermentation.a raw material for fermentation.

AntibioticsAntibioticsPenicillin by Penicillin by Penicillium chrysogenumPenicillium chrysogenum

Now most of the antibiotics were discover from soil Now most of the antibiotics were discover from soil microorganisms especially in microorganisms especially in Streptomyces spp.Streptomyces spp.

Engineered genes into production strains.Engineered genes into production strains.

Modification of existing antibiotics.Modification of existing antibiotics.

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Streptomycin from Streptomyces griseus

AlkaloidsAlkaloidsNitrogen containing organic compounds that Nitrogen containing organic compounds that derived from plants derived from plants Have medicinal purposes:Have medicinal purposes:

Atropine Atropine –– dilates pupils of the eyes (some muscle dilates pupils of the eyes (some muscle disease)disease)Morphine and codeine Morphine and codeine –– relief of painrelief of painCocaine Cocaine –– local anestheticlocal anestheticQuinine, caffeine, nicotine, strychnine, serotonin , and Quinine, caffeine, nicotine, strychnine, serotonin , and LSDLSD

Atropineserotonin

SteroidsSteroidsTypes of products Types of products include cortisone include cortisone and reproductive and reproductive hormones hormones

Human proteinsHuman proteins

Biotech DrugsBiotech DrugsMainly recombinants proteins and monoclonal Mainly recombinants proteins and monoclonal antibodies.antibodies.Highly active fields of biotechHighly active fields of biotechLots of new products each year Lots of new products each year

For list of product approve in US For list of product approve in US –– www.bio.orgwww.bio.org

Timeline in drug developmentTimeline in drug development

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12 Industrial fermentation - Shroomery