microbio lecture 21 (food microbio)

porphyra

• ____ → nori

• made from red algae

• seaweed are marine algae

spirulina

• food additive

• a spiral-shaped cyanobacteria that is used to add protein, vitamin B12 and minerals to foods

porphyra

• ____ → nori

• made from red algae

• seaweed are marine algae

spirulina

• food additive

• a spiral-shaped cyanobacteria that is used to add protein, vitamin B12 and minerals to foods

anaerobic fermentation

is used to process a wide variety of foods

acid fermentation

produces organic acid fermentation products, like lactic acid in cheese

alkaline fermentation

produces basic fermentation products, like ammonia in natto

ethanolic fermentation

produces ethanol and carbon dioxide like beer

food fermentation

preservation, improved digestibility, tastes good

why do we ferment foods?

preservation

a reason why we ferment foods:

• lactobacilli (and some other groups of bacteria) metabolism produces carboxylic acids, ammonia, and/or alcohol as waste products

• these waste products increase food shelf life

improved digestibility

a reason why we ferment foods:

• microbes break down molecules we cannot, increasing a food’s nutritional value

wild

how do we ferment foods?

• home and small-scale fermentation relies on ___ microbes, microbes found naturally in association with the food or starter cultures

• commercial fermentation use engineered microbial strains

acid fermentation

caseins, rennet

cheese production:

• making cheese starts with making curds from milk

• milk is acidic (~pH 6.6)

• Lactobacillus and streptococcus ferment lactose to lactic acid, acidifying the milk

• as milk acidifies, _____ (a hydrophobic protein) destabilize and begin to clump together

• initial result is yogurt

• _____ or another protease is added to continue breaking down casein OR heat is added to denature casein

  • soft cheeses, like cottage cheese do not have this added

denaturing caseins

• acid and heat coagulation causes small clumps of caseins

  • ricotta, cottage, cream cheese

• adding rennets = enzymatic coagulation which denatures caseine K to paracaseine K = larger hard clumps

  • swiss, muenster, cheddar

cheese production

• ferment the milk

• cheddar/cut the curd

• shape the curd

• brine/ripen

soft, semihard, hard, brined, mold-ripened

• ___ cheeses - high water content, no aging or ripening

• ___ cheeses - moderate water content, aged for several months

• ___ cheeses - low water content, aged for months or years

• ___ cheeses - salt is added

• ___ cheeses - mold spores are added

mesophilic culture

these cultures are used to make a variety of low temperature and fresh cheese. optimal growth temperature is from 86-93 F

thermophilic culture

these cultures are used to make a variety of drier and longer aged cheese. optimal growth temperature is from 95-116 F

lactic acid, propionic acid

why does swiss cheese have holes?

• sometimes, cheese goes through two rounds of fermentation

• primary ____ ____ fermentation

  • lactobacillus ferments lactose to lactate (lactic acid)

• secondary ____ _____ fermentation

  • Propionibacterium freudenreichii ferments lactate to propionate, producing CO2 as a biproduct

phytate

• Soybeans are high in protein, but also have compounds that make them difficult to digest

  • ____ chelates iron, decreasing intestinal absorption

  • Soybean lectins cause stomach upset

  • Soy protease inhibitors inhibit digestive enzymes that break down protein

• Fermentation can decrease the concentration of these harmful compounds while keeping the protein content high

soy fermentation

• A common bread mold, Rhizopus oligosporus, is used to produce tempeh

• Rhizopus breaks soy proteins down into smaller peptides and amino acids that are easily digested

• Lactic acid bacteria initially ferment soybeans while they are soaked overnight

• This creates the condition for Rhizopus to flourish

cabbage fermentation

• sauerkraut and kimchi

  • heterolactic fermentation by Leuconostoc mesenteroides

homolactic vs heterolactic fermentation

Yeast, lactic acid, oxygen

Cocoa bean fermentation:

Making chocolate from cocoa beans requires 3 successive fermentation steps, each using a different microbe

• Cocoa pulp is initially fermented anaerobically by ____ (Candida, Kloeckera, and Caccharomyces)

  • This liquifies the pulp, consumes citric acid and sugars found naturally in the beans, and produces ethanol and acetate

• As the pH increases (from 3.6 -> 4.2), ___ ___ bacteria (Lactobacillus plantarum) contribute to sugar fermentation

• Finally, _____ is introduced to the system and acetic acid bacteria (Acetobacter) consume ethanol and acids, producing CO2

yeasts, lactate and acetate, ethanol

cocoa bean fermentation:

1. _____ degrade pectin; ferment sugars and citrate to ethanol and acetate

2. Lactobacillus ferments sugars to ____ and _____

3. acetobacter oxidizes _____

bacillus

• • In contrast to lactic acid fermentation (and other acid fermentation methods), fermentation with ____ increases pH

• aerobic bacteria that are pros at proteolysis and amino acid decomposition

natto

• Soybeans are fermented using Bacillus natto to increase digestibility

• In addition to producing ammonia (which is lost as gas), B. natto produce a stringy extracellular matrix called polyglutamate

pidan

• Century eggs are duck eggs that have been treated with tea, lime (CaO), and sodium carbonate (Na2CO2 ) to raise pH and then fermented with Bacillus underground

yeast

• Saccharomyces cerevisiae (____) ferments pyruvic acid to ethanol and carbon dioxide:

  • 𝐶3𝐻4𝑂3 → 𝐶𝐻3𝐶𝐻2𝑂𝐻 + 𝐶𝑂2

• This is the key to producing leavened bread and alcoholic vegetables

injera

Yeasted or leavened breads are produced by:

• Introducing a starter yeast culture (baker’s yeast or sourdough starter) into the dough

• Kneading to develop the gluten, which allows the air pockets to room

• Waiting for the bread to rise as CO2 is produced

• _____ is a bread that used prolonged fermentation

• Teff (a high protein, gluten-free grain) cannot rise the way wheat can

• Injera is produced by fermenting teff flour with Candida for 3 days

beer

• produced by fermenting germinating barley with yeast (Saccharomyces cerevisiae)

wine

• similar to beer production, but the starting food is fruit

• Grapes (or other fruit) are fermented by yeast, most commonly Kloeckera and Hanseniaspora

Proteolytic enzymes, Oxidation, microbes

How food spoils:

Enzymatic processes

• an animal’s own _____ _____ tenderize its flesh

• Sugars in plants get converted to starch after harvest

Chemical reactions

• _____ is a common reason for food spoilage – lipid oxidation makes food rancid

Microbial processes

• _____ colonize foods and consume them – the metabolic products spoil food

water, carbohydrates

Why does meat and and fish spoil faster than fruits and veggies?

• Their nutrient composition and water content differ

  • Low ____ content -> longer shelf life

  • More ______ -> fermentation, not putrefaction

• Plant pathogens and commensal bacteria are not usually harmful to humans

food-borne pathogen sources

bacterial growth

keeping food safe relies of slowing or controlling ___ _____ and/or colonization

methods:

• Chemical preservation with spices

• Heat treatment via cooking

• Fermentation

• Dehydration and freeze-drying (no water = no microbial growth)

• Refrigeration and freezing (slows microbial growth)

• Acid treatment (adding citric acid to slow microbial growth)

• Vacuum sealing (limits oxygen)

• Pasteurization (short high heat treatment kills bacteria)

• Chemical additives

bioprospecting

the search for organisms with potential commercial applications

• The goal is to find microbes (or other organisms) that naturally produce a useful product

• We then learn how the product is produced and scale up production

good microbial products

• Genetically stable and able to be manipulated

• Inexpensive to grow

• Safe

• Efficiently produce the desired product

• Amenable to an easy harvest mechanism

Common species used to create microbial products include Escherichia coli, Bacillus subtilis, Candida utilis, and Aspergillus niger

microbial vitamins

• Many essential vitamins are highly complex molecules

• B12 is both very complex and important for red blood cell formation

• We make most commercially available B12 using bacteria

b. thurinigenesis

• ____ ______ is the most commonly used biological pesticide worldwide

• ____ ______ produces Bt toxin, which is fatal to caterpillars

• Bt toxin binds to the intestinal wall, lyses the epithelial cells, allowing normal gut bacteria to colonize the rest of the caterpillar body

bioplastics

• An ancient way of storing carbon

• Similar to lipid droplets only solid at ambient temperature

• Two major kinds

  • Polyhydroxybuturate (PHB)

  • Polyhydroxyalkanoate (PHA)

• Produced by many different bacteria and archaea

• Can be used to generate large amounts of biodegradeable plastic!

• Technology has been in development for decade

bioremediation

• the cleanup of oil or other pollutants by microorganisms

• examples include cleanup of oil spills, pesticides, dechlorination, solid wastes, and sewage treatment

catabolizing

• One of the ways microbes gain energy is by ______ molecules

  • they see “waste” products as “food”

• Microbes have evolved to extract energy (in the form of electron transfer) from a diverse range of organic and inorganic molecules

• This allows microbes to play key roles in the circular economy

anaerobic bioremediation

• Chlorinated Solvents

• Tetrachloroethene, trichloroethene, trichloroethane, and carbon tetrachloride

• Hazardous and not easily degraded

• Anaerobic degradation sequentially removes chloride ions

hydrocarbon decomposition

• A variety of naturally occurring bacteria, fungi and green algae are able to oxidize hydrocarbons

• Hydrocarbon-oxidizing bacteria develop on oil films and slicks

• Hydrocarbon-oxidizing bacteria associate with oil droplets and concentrate at the oil water interface

• The bacteria oxidize the oil to CO2

• Studies of oil spills indicate that the bacteria increase in number 103 -106 times shortly after the spill

• Under ideal conditions the microbes can oxidize ~80% of the nonvolatile components within 1 year

• These microbes are good enough at their job that antimicrobial agents need to be added to gas storage tanks to prevent degradation of the fuel at oil-water interfaces

phosphates and nitrogens

hydrocarbon decomposition

• The addition of nutrients like ______ and _____ to the spill area increases bioremediation rates

• Although microbes are a considerable aid, big spills still require human efforts to ensure a timely cleanup

D. radiodurans

radioactive bioremediation:

• A lethal level of radiation for humans is about 700 rads.

• ______ _______ can withstand 1.5 million rads

• Exposure to high levels of ionizing or UV radiation lead to extensive DNA damage

• In contrast to other bacteria, ___ _____ can repair its DNA

ddrA

How does D. radiodurans survive such high levels of radiation??

• Using microarrays, Harris et al identified a single open reading frame whose expression was induced ~2-fold following treatment of D. radiodurans with ionizing radiation

• Expression of RecA, another DNA repair protein, was only increased 1.5-fold under the same conditions

• Deletion of this ORF (renamed ___) resulted in increased sensitivity to ionizing radiation

• Deletion of_______ results in increased sensitivity to DNA damage

____ protects single stranded 3’ ends from endonuclease activity

D. radiodurans for bioremediation

• The mercuric reductase gene from E. coli has been cloned into D. radiodurans to detoxify the ionic mercury found in radioactive waste generated from nuclear weapons manufacture

• This strain of D. radiodurans reduces Hg(II) to less toxic elemental mercury

• The same lab has also been able to engineer D. radiodurans to detoxify toluene as well

poly

Ideonella sakaiensis breaks down ____(ethylene terephthalate), a component of plastics

e-waste

• Methylobacterium extorquens can be used to extract rare earth metals from e-waste

• M. extorquens grows on and sequesters lanthanides and other rare metals

wastewater treatment

• Wastewater is the liquid extracted from sewage and industrial sources

• It contains both inorganic and organic components including pathogenic bacteria, phosphates, and trace metals

• 15,000 wastewater treatment plants exist in the US. Collectively they process 40 billion gal of wastewater a day

primary treatment of wastewater

consists of removal of solids by passing it through a series of screens and letting the effluent stand for several hours to let the solids settle to the bottom

secondary treatment of wastewater

employs microbes to reduce the organic load of the wastewater to environmentally acceptable levels.

tertiary treatment of wastewater

uses chemical methods (precipitation, filtration, and chlorination) to remove inorganics like phosphate from wastewater (Very Expensive and not always done)

anoxic secondary treatment

• A set of digestive and fermentative reactions carried out by a variety of bacterial and archaeal species

• The efficiency is measured in terms of the percentage decrease in biological oxygen demand (BOD)

• BOD refers to the amount of oxygen that would be consumed if all the organics in one liter of wastewater were oxidized by bacteria, archaea and protozoa

• A good wastewater treatment plant removes 95% or more of the initial BOD

• The degradation process takes place in “sludge digestors” or “bioreactors”

anoxic sludge digestor

• Macromolecules are broken down into soluble components (monosaccharides, amino acids, fatty acids)

• These are broken down into acetate, H2 , and CO2 -- substrates for methanogenic bacteria

• The major products of anoxic sewage treatment are CO2 and methane

  • The methane is either burned off or collected for use in electric generators to heat and cool the treatment plant

aerobic sewage treatment

Trickling filter method

• The wastewater is sprayed onto a 2m deep bed of crushed rocks

• As the liquid slowly trickles through the bed the organic matter adsorbs to the rocks allowing for microbial growth on the surface

• The microbes mineralize the organic matter to CO2 , NH4 , nitrate, sulfate, and phosphate

Activated sludge method

• The wastewater is mixed and aerated in a large tank

• Slime forming bacteria (Zoogloea ramigera for example) grow and form flocs (slime encased microcolonies)

• The flocs attract protozoa, other small animals, and some bacteria and fungi

• The effluent containing the flocs is pumped to a clarifier where the flocs settle. Some is returned to the incubator to serve as an innoculum. The rest is sent to a sludge digestor

→ The wastewater spends only 5-10 hours in an activated sludge tank, not enough time for complete oxidation of organic materials

→ Adsorption to the floc is the primary means of reducing BOD in the liquid. Main BOD reduction occurs after transfer of the floc to the sludge digestor

trickling filter method (aerobic sewage)

• The wastewater is sprayed onto a 2m deep bed of crushed rocks

• As the liquid slowly trickles through the bed the organic matter adsorbs to the rocks allowing for microbial growth on the surface

• The microbes mineralize the organic matter to CO2 , NH4 , nitrate, sulfate, and phosphate

activated sludge method (aerobic sewage)

• The wastewater is mixed and aerated in a large tank

• Slime forming bacteria (Zoogloea ramigera for example) grow and form flocs (slime encased microcolonies)

• The flocs attract protozoa, other small animals, and some bacteria and fungi

• The effluent containing the flocs is pumped to a clarifier where the flocs settle. Some is returned to the incubator to serve as an innoculum. The rest is sent to a sludge digestor

these systems are:

• Energy intensive

• Remove nitrogen from the water cycle (and release it in the form of N2O, contributing to global warming)

• Rarely produce much, if any, potable water

Thermophilic sludge digestion

adding heat to the anaerobic digestion step

• Pros: doubles or triples energy recovery by producing more biogas

• Cons: more nutrients are released, complicating tertiary treatment

microbial hacks to wastewater treatment

• The digestion of sludge (the solids) is carried out by microbes – we can tune which microbes are there

• Develop microbial strategies to make treatment more efficient

microbial circular economy

Highlighting the role of microbes in the circular economy

→ Reduce, reuse, recycle at a microbial scale

circular economy

The circular economy is a model of production and consumption, which involves sharing, leasing, reusing, repairing, refurbishing and recycling existing materials and products as long as possible. In this way, the life cycle of products is extended.