Microbiology Exam 3

Food Microbiology is a mix of…..

“kill pathogenic microbes” and “encourage growth of useful microbes”

What is used in canning to sterilize food?

High pressure and High temperature

-Commercial canning is done under pressure while home canning is sometimes done under pressure using a retort.

_____ is a major method of food preservation

Canning

Commercial Canning Process

1. Blanching in hot water or steam denatures enzymes that might alter color, texture, or flavor.

2. Can are filled to capacity, leaving minimal dead space

3. Steam is used to exhaust, or drive out, dissolved air

4. The cans are sealed

5. Cans are sterilized by pressurized steam in a retort, similar to an autoclave

6. Cans are then cooled by submersion in a water bath or by spraying them with water

Endospore-Forming Microbes

-Not all are killed

-obligate thermophiles that don’t grow at normal temperatures

Thermophilic anaerobic spoilage

Cans left at a high temperatures and spoil due to the growth of _______.

Flat sour spoilage 

Spoilage with no gas production. 

Mesophilic Microbes

Spoil if food is not processed well enough and/or if there is contamination after processing-canner didn’t get hot enough, microbes got in.

Aseptic Packaging

Sterilized packaging is filled with liquid foods

Radiation

X-rays or gamma radiation to sterilize foods 

-different doses will have different effects 

High-Pressure food Preservation

Submerge in tanks of pressurized water

-reached up to 87,000 psi

-Kills many pathogenic bacteria and non-pathogenic bacteria that shorten shelf life

Food Production-Cheese 

-Separate the curd (made of the protein casein after processing by the enzyme rennin) from whey 

-The curd is ripened by various lactic acid producing microbes

-Cheddar and Swiss-various lactic acid bacteria 

-Propionibacterium species forms CO2 that forms the holes in Swiss cheese

-Cambert, Blue and Roquefort cheeses are ripened by Penicillium molds

Yogurt Production 

-Thickened milk inoculated with various microbes

-Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, and Lactobacillus case

Butter/buttermilk production-The aroma and flavor is from diacetyls from lactic acid bacteria 

Kefir and Kumiss-lactic acid and lactose fermenting yeasts

Cacao, Beer, Wine, Breadmaking Production

____-fermented

_______-Saccharomyces cerevisiae

___and______-made anaerobically so yeasts ferment sugars into alcohol

_______ yeasts are encouraged to work aerobically to produce the air pockets in bread.

-lactic acid bacteria are also involved in rye and sourdough for their distinctive flavors

Barley, Lager yeast and ale yeast, Sake, Wine Production

___-malted to form amylases and the malt is extracted

______-faster and forms clumps that float due to CO2

______-slower and sinks 

_____-unmalted rice, uses Aspergillus to convert starch to sugars

_____-made from grapes

-uses lactic acid to reduce the amount of malic acid 

Acetobacter and Gluconobacter convert ethanol (apple cider also) to acetic acid to turn wine into vinegar

Winemaking Process

1. Grapes are tested and picked 

2. Grapes are crushed and destemmed 

3. Sulfite is added to kill undesirable yeasts and bacteria 

4. Yeast inoculum is added 

5. Fermentation occur

6. Result is pressed to separate solids from wine 

7. Wine is clarified in settling vats 

8. Wine is filtered 

9.  Wine is aged 

10. Wine is bottled 

Industrial Fermentation

Large scale cultivation of bacteria to produce stuff

-biotechnology comes into play a lot with this

Bioreactor

A large vessel where aeration, temperature, pH and nutrients can be closely monitored so microbes can produce products.

Batch production

all harvested at once

Continuous flow production

Some is removed at a time

Primary metabolite

formed at the same time as new cells (in log phase)

Secondary metabolite 

formed after the logarithmic growth phase, in the stationary phase 

Metabolomics

Study of all the cellular processes using chemical fingerprints of cellular metabolism 

Strain

differs from others of the same species in a significant way

-contains or lacks a different enzyme 

____improvement is a big thing-mutagenesis 

site-directed mutagenesis

Allows engineers to change specific DNA bases to modify a single amino acid if necessary

Amino Acids and Citric Acids

-Glutamic acid for monosodium glutamate  (MSG)

-Lysine and Methionine for feed additives

-Phenylalanine and aspartic acids as ingredients for aspartame

Enzymes 

•Amylases for corn syrup, paper sizing (like textbook pages), glucose from starch

-The first biotechnology patent in the USA was for microbiological production of amylase

•Glucose isomerase – convert glucose to fructose

•Proteases – reduce amount of gluten in wheat, meat tenderizers, detergents

•Rennin – for cheesemaking, previous from fungi, now from genetically modified bacteria

Antibiotics

all originally microbial products, still any are

-Streptomyces hygroscopius – different strains make almost 200 antibiotics

Steroids, estrogen, and progesterone

are all difficult to isolate from animal sources or chemically synthesize, but easy for microbes to make

Vaccines

•Antiviral vaccines in chicken eggs or cell cultures

•Bacterial usually requires large quantities of the bacteria (attenuated or not)

•Vaccines often consist of polysaccharides from the bacteria

•Recombinant DNA technology for subunit vaccines – contain only antigenic fragments that stimulate the best immune response

•HepB vaccine

Microbes as Products

•Saccharomyces cerevisiae is an industrial product on its own

-Used by bakers and brewers

•Rhizobium and Bradyrhizobium to inoculate legumes for nodule growth

-Part of the nitrogen cycle – nodules fix nitrogen from N2 to NO3

•Bacillus thuringiensis is used as a natural insect control-used in farming

-Produces the insecticidal Bt-toxin

-Used in farming and in mosquito control

Energy Sources

•Microbes convert biomass to energy sources (bioconversion)

•Methane – common and convenient bioconversion energy source

•Ethanol – common biofuel, used as a fuel additive

-E10 (90% gasoline, 10% ethanol) is very common, despite its deficiencies

-Needs special transportation due to water absorption and is less energy dense.

•Other biofuels derived from cellulose have more interest now

-But it’s harder and more expensive

•Algae sourced oil and other biofuels is theoretically attractive

-Biodisel and possibly jet fuel

-Has not been put into mass production due to various drawbacks and cost

Biotechnology

•The use of organisms/cell components to make a product

•Recombinant DNA is a natural occurrence in microbes

•Artificially recombining DNA was developed in the 1970s and 1980s

•Often used for inserting DNA into a new organism for the purpose of generating a product

Recombinant DNA

Recombinant DNA Information

•A vector is a DNA molecule used to insert foreign DNA into a cell

-Must be self-replicating – plasmid or viral genome

•Insert a gene of interest into the vector

Introduce the vector into the cell – cell produces the product

-Sometimes the product is what the gene codes for

-Sometimes the product is the plasmid itself!

Biotechnology Tools-Artificial Selection

•Yeast strains can be selected for better tasting beer, or tolerating the higher alcohol content of wine

•Antibiotic producing strain have been selected for

Biotechnology Tools-Mutations

•Penicillium was randomly mutated using radiation – the strains with the highest amount of penicillin produced were selected

Biotechnology Tools-Site Directed Mutagenesis

modify a single amino acid in a gene to change the protein

Restriction Enzymes

•Endoenzymes that form breaks in DNA at specific sequences

•Originally observed in phage defense

-If a phage infected a bacterial cell with the correct restriction enzymes, the restriction enzymes would cut apart (digest) it’s DNA

-Bacterial DNA was protected by methylating the cytosines in the genome, preventing digestion

•Named for the bacteria it was originally found in

Blunt Ends

Ends where both strands of DNA are broke at the same base

AGTG| |CACT

TCAC| |GTGA

Sticky ends

•the DNA strands are broken at different bases, leaving short segments of ssDNA-offset

GTGCA|C

C|ACGTG

•can be used for combining DNA from multiple sources

-DNA ligase will rejoin the sticky ends

Restriction Enzymes

Vectors

-Must be self replicating in a cell and also be large enough to manipulate outside of the cell

-Most are bacterial plasmids

—Origin of replication

—R gene for selection

—Restriction enzyme sites

-Viral genomes can also act as _____

-Shuttle _____ can exist in multiple species

What does PCR stand for?

Polymerase Chain Reaction

Thermus aquaticus

•thermophilic bacterium that has a DNA polymerase that can survive the melting temperature of DNA

PCR

•PCR is very useful, but has limits

-Can only amplify shorter sequences – usually sub 10kb

-With optimized polymerases, can hit 30kb

•Can be used for diagnostics

-Amplify using primers specific to a specific species or strain, gives a yes/no answer fast

Real-time PCR/Quantitative PCR

•a method for quantifying DNA after replication using fluorescent dyes

-Method 1: tag dsDNA with fluorescent dyes

-Method 2: DNA polymerase breaks down probes that contain a fluorescent dye and a quencher

-This quantification is often used for diagnostics

Reverse transcriptase PCR

•uses a reverse transcriptase from a retrovirus to make DNA from RNA for further amplification

-Used to diagnose viruses with RNA genomes (often combined with RT-PCR)

-Also used to get intron-less genes from eukaryotes

ddPCR

•Digital Droplet PCR. Like qPCR but tiny and more accurate.

Gel Electrophoresis

•Agarose (different than agar) is solidified in a slab

•DNA is inserted

•An electric current is used to pull the DNA from the start to the end

•Due to the network of agarose, smaller DNA fragments move faster than larger DNA fragments

Putting it all together pt 1

•Insert plasmid into bacteria – transformation

-Chemically competent cells – chemically induced to allow the cell to take up foreign DNA

-Electroporation – using an electrical current to form pores in the cell membrane, allowing foreign DNA to enter

—Must be protoplasts – no cell walls

-Gene gun – exactly what it says on the tin

—Use DNA coated gold micro-beads and fire them at the cells – often used in plants

Putting it all together ot 2

•Select colonies or cells with the foreign DNA in them.

-Antibiotic resistance is the first screen – often ampicillin or kanamycin

-A second screen is often blue-white screening

-lacZ will convert X-gal to a blue pigment – by inserting the gene of interest into lacZ, it’s possible to tell what microbes have the gene of interest.

Putting it all together 

1995

First free-living cell-Haemophilus influenzae

1977

First genome sequenced-bacteriophage

2003

First human genome completed-started in 1990

Shotgun sequencing

sequencing of small, random pieces of DNA, that are then assembled by a computer

Bioinformatics

Field of genomics(ish) specifically via computer-assisted analysis

DNA Sequencing-Shotgun

Genomics

Study of all the genes in a genome

Proteomics

Study of all proteins in a cell

Transcriptomics

Study of how genes are transcribed

Metabolomics

Study of the metabolism in a cell

Metagenomics

•sequencing and studying of DNA from environmental sources

-Only way some microbes have been catalogued!

Biotechnology – Single Cell Sequencing

•Often studying the transcriptome of a single cell

•Used for studying diseases and stuff

•Really interesting

•Not really relevant to microbes

Agricultural Use

•Often, a gene gun is used to introduce recombinant DNA

•Agrobacterium-mediated transformation is much more elegant

•Agrobacterium tumefaciens inserts DNA on a Ti plasmid (tumor inducing plasmid) and the DNA incorporates into the genome of the plant

•In the wild, A. tumefaciens only infects eudicot plants – flowering dicot plants

•In the lab, we can use the chemical acetosyringone to induce A. tumefaciens to inject DNA into anything we want

•Ask how I know.

Agrobacterium Mediated Transformation

Ethical Issues

•Gene escape – especially in cases of resistance or pathogenicity

•Bt toxin and modified plants have been shown to kill bees and monarch caterpillars that consume Bt-toxin laced pollen

•Genetically modified crops resistant to herbicide have lead to massive amounts of herbicide resistance in weed species.

•Genetic testing and counselling – when is it ethical? Who should have access to the information?

Microbes live in a variety of habitats because of their abilities to

Use a variety of carbon and energy sources

Grow under different physical conditions

Extremophiles

live in extreme environments

ph

temperature

salinity

Symbiosis

Two differing organisms living together in close association that is beneficial to one or both of them

example: Ruminants (such as sheep and cows) and the bacteria in the rumen

Mycorrhizae (example of animal-microbe symbiosis)

contribute to plant growth

fungus root

-Endomycorrhizae – form vesicles (smooth oval bodies) and arbuscules (tiny bushlike structures inside plant cells) - arbuscular

-Ectomycorrhizae – form mycelial mantle over the small roots of trees (mainly pine and oak).

Mycorrhizae

Fungi living in close association with plant roots

Extend surface area of roots and increase the absorption of nutrients, especially phosphorous

Plants can be very reliant on these mycorrhizae

Biogeochemical Cycles

-1 gram of soil may contain up to 1 billion bacteria

-1 milliliter of ocean water can contain up to 1 million bacteria.

-Involved in the recycling (oxidation and reduction) of chemical elements

-Many are symbionts of plants (rhizobia)

-Microbes in soil metabolize organic matter, form detritus (non-living particulate organic matter).

-Saprotrophic bacteria and fungi

Factors affecting microbial abundance in soils

Moisture content

-Moist soils support microbial growth and diversity better than dry soils

Oxygen

-Oxygen dissolves poorly in water

-Moist soils are lower in oxygen than dry soils

-Anaerobes predominate in waterlogged soils

pH

-Highly acidic and highly basic soils favor fungi

Temperature

-Most soil organisms are mesophiles

-Live well in areas without extreme summer or winter

Nutrient availability

-Most soil microbes utilize organic matter

-Microbial community size determined by how much organic material is available

Microbial populations present in the soil

-Bacteria are numerous and found in all soil layers

-Archaea present but are difficult to culture and study

-Fungi are also populous group of microorganisms

-Animal viruses are rarely found free in soil; bacteriophage are highly abundant in soil

-Algae live on or near the soil surface

-Most protozoa require oxygen and remain in the topsoil

Microbes perform a number of functions

-Cycle elements and convert them to usable form

-Degrade dead organisms and their wastes

-Produce compounds with potential human uses

The Carbon Cycle

-The primary biogeochemical cycle

-Carbon fixation – any process in which gaseous carbon dioxide is converted into a solid organic compound.

-Photosynthetic fixation – photoautotrophs

The return of CO₂ to the atmosphere by respiration closely balances its removal by fixation. However, the burning of fossil fuels adds more CO₂ resulting in a steady increase.

The Nitrogen Cycle

-Molecular nitrogen (N₂) makes up almost 80% of the atmosphere

-For organisms to assimilate and use nitrogen it must be fixed – taken up and combined into organic compounds.

—Ammonification

—Fixation

—Nitrification

—Denitrification

The Nitrogen Cycle-Ammonification

The Nitrogen Cycle-Nitrogen

The Nitrogen Cycle-Denitrification

The Nitrogen Cycle-Nitrogen Fixation

Nitrogen Fixation-Rhizosphere

-Cyanobacteria: heterocysts (a specialized structure carrying nitrogenase enzymes)

-Azotobacter

-Beijerinckia

-Clostridium pasteurianum

Nitrogen Fixation-Lichens

-symbiotic relationship between a fungus and:

—Cyanobacteria

—Algae

Nitrogen Fixation-Root Nodules

Species invade and form bacteria filled nodules on the roots

-Rhizobium

-Bradyrhizobium

-Frankia

The Sulfur Cycle

Seawater Microbiota

-Divides into zones based on light and O2 availability

-Includes abyssal zone dominated by archea

—Phytoplankton (forms basis of oceanic food chain)

Chemical Water Pollution

-Resistant chemicals may be concentrated in the aquatic food chain

-Mercury is metabolized by certain bacteria into a soluble compound, which is concentrated in animals

-Microbes can also be used to detoxify or degrade pollutants – bioremediation

—Oil spills, municipal solid waste

Microbial Water Pollution

-Microbes are filtered from water that percolates into groundwater

-Some pathogens are transmitted to humans in drinking and recreational water

-With the addition of nutrients, these can be large problems.

Eutrophication

-Overabundance of nutrients in lakes and streams

-Caused by

—Addition of organic matter

—Or inorganic matter

—.Phosphates

—.Nitrogen

—.Which cause algal blooms – red tides

Biochemical Oxygen Demand (BOD)

-Bacterial decomposition of organic matter uses up O₂ in water.

-Biochemical oxygen demand or B.O.D. is the amount of dissolved oxygen needed by aerobic biological organisms in a body of water to break down organic material present in a given water sample at certain temperature over a specific time period.

Water Purity Tests-Indicator organisms

-Used to detect fecal contamination

-Coliforms

-Enterococcus

Water Purity Tests-Most Probable number

/100 ml of water

-One of the indirect methods for getting the concentrations of bacteria in a sample

Coliforms

-Aerobic or facultatively anaerobic, gram-negative, non–endospore forming rods that ferment lactose to acid plus gas within 48 hours, at 35^oC

Municipal Water Purification Treatment

Primary treatment

Settling and screening of solids – primary sludge

Secondary treatment

-Removal of much of the BOD

-Disinfection (kind of)

-Water can be used for irrigation

Tertiary treatment (if applicable)

-Removal of remaining BOD, N, and P

-Disinfection

-Water is drinkable