Nutrition and Growth:• Nutritional Classification

– Energy source– Carbon source

• Requirements for Growth– Temperature - pH– Salts - Oxygen– Nutrients - Light

• Laboratory Cultivation– Media Types– Growth Curves– Measuring Growth

Nutritional Classification:

What does it mean to use water to reduce carbon dioxide? How does this relate to oxygen generation or not?

This is us!

Carbon Source: CO2 Only

• Autotrophs don’t feed on organic matter to acquire the small organic compounds needed for cell growth.

• Instead they use energy and electrons to reduce 6 carbon dioxide to 1 glucose, which is then converted to all the small organic compounds needed for cell growth. They do it all themselves; hence “auto”.

• The metabolic pathway responsible for “CO2 fixation” is the Calvin Benson Cycle.

6 cycles for the 2 GP needed to make 1 glucose.

Reduction step

(adding e-)

Light for Energy: Phototrophy

• Light excites chlorophyll (photopigment) electrons to a higher energy state.

• As with electrons from NADH in respiration; these light excited electrons transport through an ETC; energy released pumps protons across a membrane creating PMF.

• Like in oxidative phosphorylation of respiration, the PMF drives ATP synthesis by phosphorylating ADP; because light and not NADH oxidation is the source of excited electrons, we call ATP production by phototrophy, photophosphorylation.

This is called Cyclic Photophosphorylation; because electrons cycle back to chlorophyll.

Photoheterotrophs do this!

Non-cyclic Photophosphorylation:Excited electrons leave chlorophyll and end up in the form of the electron carrier, NADPH; chlorophyll needs to get new electrons from somewhere.

Electrons can come from water results in O2 production. All plants, algae, and cyanobacteria do this; oxygenic photosynthesis.

Some bacteria can not tolerate oxygen, nor can they get electrons from water to supply chlorophyll. They may use other compounds like H2S; anoxygenic photosynthesis.

Requirements for Growth:• An organism will grow and reproduce when it’s in an

environment where it can tolerate all physical and chemical conditions. (Shelford’s Law of Tolerance)– E.g. nutrients, oxygen, salts, light and pH levels are optimum for

growth, but if temperature is above that cell’s tolerance range it won’t grow.

– What might you conclude if you could not isolate a marine bacterium on agar that was made without salts, but all other conditions were optimum?

• Growth will be slower when one or more condition is not optimum, yet within the range of tolerance.

• Different microbes have different tolerance ranges for different physical and chemical conditions.

Temperature: Peaks in these plots represent the optimum growth temperature for that group of temperature tolerance. Growth rate decreases at temperature values higher and lower than the optimum until there is no growth (maximum and minimum growth temperatures).

Food Storage Considerations15-50ºC = Danger Zone

Many psychrotrophs are involved in food storage, even at refrigerator temperatures (4ºC).

Rate of cooling will be different depending on the surface to volume ratio of your stored food.

Osmotic Pressure Tolerance

• Hypertonic (solute concentration higher outside of cell) can cause plasmolysis.

• Some bacteria are osmophiles; if the solute is salt we call these halophiles.

• Some bacteria are more osmotolerant than others (make compatible solutes).

• pH:– Acidophiles (pH 0 – 5) acid mine drainage, vinegar– Neutrophiles (pH 5.5 – 8) pure water, blood– Alkalophiles (pH 8.5 – 10.5): soap, ammonia

• Light:– Visible Light (380-760 nm; phototrophs need it)– Ultraviolet Light (130-380nm; DNA damage)

• Nutrients:– Carbon (quality of organics for heterotrophs)– Macronutrients (nitrogen, phosphorous, sulfur)– Trace elements (micronutrients; iron, copper, zinc…)

Oxygen Requirements / Tolerance

Anaerobic Growth Techniques:

Media Types• Complex versus Defined:

– When every chemical and its amount in the media is known, we call it a defined media.

– When one or more ingredient is of uncertain composition (e.g. yeast extract), we call this an undefined or complex media.

• Non-selective versus Selective:– Media designed to favor the growth of as many different bacteria as

possible is called a non-selective media.– Media with special ingredients or sources of organic matter to enrich the

growth of a specific group of bacteria over others is called a selective media.

– (E.g. lactose broth for coliform bacteria enrichment).

• Differential: – Often these are selective media in the solid (agar) form that have had

additional ingredients added so particular kinds of selected bacteria can be differentiated from other growing bacteria.

– (E.g. coliform bacteria on ENDO agar will be a darker red color with a metalic sheen to the colonies, as compared to other bacteria on the same media.)

Prokaryote Exponential Growth (N = 2n)

• Every time bacteria in a culture divide by binary fission the population number increases 2-fold.

• The time it takes for the bacteria to divide, and double the population, is called the generation time.

• With each generation (n) the total population (N) increases by a power of 2.

• Intrinsic growth rate (µ) is the number of generations per hour (h-1).

Plotting Growth on a Logarithmic Scale:

Bacterial Growth Curve(For a “closed” culture; e.g. a tube of broth media)

Growth Curve Phase Summary:• Lag Phase:

– Bacteria make adjustments to new conditions.– Takes time to “express genes” for new protein types (like enzymes)

now needed.

• Log Phase:– Physiologically adjust for maximum growth for the environmental

conditions (note log scale in previous growth curve plot).

• Stationary Phase:– Death and growth balance, i.e. they are in equilibrium.– Growth slows due to some resource becoming limiting (used up).– Growth may also slow due to a change in the environment that

exceeds the bacterium’s tolerance range (e.g., pH decrease).

• Death Phase:– Conditions promote death faster than growth.– Death rate or decline is logarithmic; more and more cells die for

each hour of decline (note log scale in previous growth curve plot).

Viable Plate Counts:

• There are two methods for evenly spreading bacteria across an agar plate for counting:

- Pour Plate (We did this!)

- Spread Plate

• Each method requires a series of quantitative dilutions to reduce cell density so that we can see well isolated (separated) colony forming units (CFU) for counting.

• Here they performed a series of 1:10 dilutions and plated the same volume (1 mL) using the pour plate technique.

• After incubation, look for the plate with between 30 to 300 CFU; count it.

• Bacteria Density = (CFU * dilution factor) = 32 CFU * 10,000/mL = 320,000 CFU/mL

Direct Microscopy CountOnly works well for very large cells; not very good

for most bacteria!

Growth Monitored by Turbidity

This is a rapid assay for broth culture growth, as is does not require incubation.

How can you convert turbidity to bacteria cell number per milliliter (i.e., CFU/mL)?

Multiple Tube Test for Most Probable Numbers (MPN)

As the inoculum volume decreases, the probability of there being growth of bacteria also decreases. More positive growth tubes having received the smallest inoculum volume means a greater number of bacteria in the inoculum (sample). Every combination of growth results has a statistical probability of being due to a particular, or most probable, number of bacteria in the inoculum (sample).

Reading the MPN Table:

We will use this in lab this week to determine MPN for coliform bacteria.