Chapter 5: Culture Microbes and Measuring their Growth (5.13-5.16)
Although halophiles require at least some NaCl for growth, the NaCl optimum varies with the organism and is habitat dependent. For example, marine microorganisms typically grow best with ______________ NaCl
1-4%
____________ microbial habitats are common in nature and include muds and other sediments, bogs, marshes, water-logged soils, intestinal tracts of animals, sewage sludge, the deep subsurface of Earth, and many other environments.
Anoxic
Why is it that only 3 group of obligate anaerobes (Bacteria and Archaea, a few fungi, and a few protozoa) are known?
Because there are many habitats for anaerobes, they are very common in nature and highly diverse
Organisms that are _________ (which is a mixture of freshwater and seawater) may or may not be halophilic
Brackish waters
Why are anaerobic microorganisms inhibited in their growth or even killed by oxygen?
Molecular oxygen (O2), per se, is not toxic, but O2 can be converted to toxic oxygen by-products, and it is these that can harm or kill cells not able to deal with them. These include superoxide anion (O2 -), hydrogen peroxide (H2O2), and hydroxyl radical (OH·).
True or false: organisms from extremely hypersaline environments DO NOT require higher levels of NaCl
False: organisms from extremely hypersaline environments require higher levels of NaCl
what is widely distributed among halophilic bacteria?
Glycine betaine, an analog of the amino acid glycine
OH* + e^- + H+ ==>
H2O (water)
H2O2 + e^- + H ==> H2O
H2O + OH* (hydroxyl radical)
O2 + e^- + 2H^+ ==>
H2O2 (hydrogen peroxide)
What will happen if the cell keeps losing water?
If a cell has no strategy to counteract this, it will become dehydrated and unable to grow.
How does low aw affect our food?
It wont get spoiled by bacterium as fast. Bacteria need water in order to grow. However, foods such as dried fruit wont get spoiled (and bacteria need water, if not they dry out). That is why we store food in the refrigerator because it is an environment where it is not optimal for growth (4 degrees Celsius). Another reason why we salt our food for storage.
what solute is compatible with the compatible solutes? Where is it found
KCl, is the compatible solute of extremely halophilic Archaea, such as Halobacterium, and of few extremely halophilic bacteria
halophiles need what to survive?
NaCl (sodium chloride), which is absolute and cannot be replaced by other salts. Thus, they are salt loving (and grow best at low water availability)
O2 + e^- ==>
O2^- (superoxide)
Where do these organisms grow when thioglycolate broth is added?
Obligate aerobes grow only at the top of such tubes. Facultative organisms grow throughout the tube but grow best near the top. Microaerophiles grow near the top but not right at the top Anaerobes grow only near the bottom of the tube, where O2 cannot penetrate
Osmophiles
Organisms able to live in environments high in sugar. very sweet creatures
Matric water activity
a measure of water bound to a surface, is measured in the same way as osmotic water activity but can drop to significantly lower than 0.6 and still contain viable microbial communities
How does water diffuse?
from regions of high water concentration (low solute concentration) to regions of lower water concentration (higher solute concentration) in the process of osmosis
Thus, regardless of whether it can respire O2, an organism exposed to O2 will experience toxic forms of oxygen, and if not destroyed, these molecules can wreak _______________
havoc in cells
Microbes more are more likely to grow in
high aw
The cytoplasm of a cell typically has a ________________ solute concentration than the environment, so the tendency for water is to diffuse into the cell
higher
typically, the cytoplasm has a __________ concentration than the surrounding environment; thus, the tendency is for water to move into the cell, known as _________
higher positive water balance
changes in osmotic concentrations in the environment may affect microbial cells. What is hypertonic solution?
higher osmotic concentration water leaves the cell = cell membrane shrinks from the cell wall (and plasmolysis may occur)
what condition (hypertonic or hypotonic) do bacteria DON'T mind?
hypotonic
superoxide reductase
in some strict anaerobes, converts O2 to H2O without producing O2
extreme halophiles
organisms that require very high levels (15 percent to 30 percent) of NaCl; often unable to grow at lower concentrations
In nature, _____ _____ are of interest mainly in habitats with high concentrations of salt
osmotic effect
What is an essential nutrient for microbes? Hint: they are unable to metabolize OR grow without it
oxygen
The by-products (that were listed) are the reduced form of ________ to ______ in respiration
oxygen water
compatible solutes can
synthesize cytoplasmic solutes
Water availability not only depends on how moist or dry an environment is but is also a function of-
the concentration of solutes (salts, sugars, or other substances) dissolved in the water that is present
positive water balance. Hint: this the normal state of the cell
the cytoplasm has higher solute concentration than the environment so water diffuses into the cell
Water activity (aw)
the ratio of the vapor pressure of air in equilibrium with a substance or solution to the vapor pressure of pure water. Meaning, ratio of vapor pressure of air in equilibrium with a substance or solution to the vapor pressure of pure water
how do microbes keep toxic oxygen molecules under control?
they do this in much the same way plants and animals do. Superoxide anion and H2O2 are the most abundant toxic oxygen species, and all cells have enzymes that destroy these compounds.The enzymes catalase and peroxidase attack H2O2, forming O2 and H2O, respectively
example of reducing agent
thioglycolate broth
xerophiles
those able to grow in very dry environments (made dry by lack of water rather than by dissolved solutes)
Many aerobes are facultative, meaning that -
under the appropriate nutrient and culture conditions they can grow in the absence of O2. summary: can live with or without oxygen
what are the values of aw?
vary between 0 (no free water) and 1 (pure water)
How is water availability measured/expressed?
water activity (aw)
water activity is known as ______; expressed in physical terms
water availability
Osmosis
water diffuses from high water (low solute) to low water concentrations (high solute)
what will happen if the cell is placed in an environment where the solute concentration exceeds that of the cytoplasm?
water will flow out of the cell
How do we exclude oxygen for anaerobes?
we need reducing agents: chemicals that may be added to culture media to reduce oxygen
anoxic
without oxygen (O2 free)
From growth data obtained from extremely halophilic representatives of all three domains of life, there appears to be a common lower water activity limit for living organisms, and this limit is ________
0.61
Microorganisms that inhabit marine environments almost always show an NaCl requirement and grow optimally at the aw of seawater, _________
0.98
since halophiles grow best with high levels of salt and low water availability, their aw requirement is
0.98 (seawater)
seawater contains ___________ of sodium chloride (NaCl)
3%
How does a reducing agent work? Give an example of a reducing agent.
A reducing agent is the substance in redox reaction that donates an electron, therefor its the electron donor in the reaction
What are compatible solutes, and when and why are they needed by the cell? What is the compatible solute of Halobacterium?
Compatible solutes are typically highly soluble organic molecules that do not inhibit important cellular processes and help organisms survive osmotic stress. They are specifically needed by an organism to maintain a positive water balance when transferred from a medium with high aw to low aw. In halophilic Archaea such as Halobacterium, the compatible solute is KCl
Halotolerant are microbes that ____________ require salt but can grow in ________________
DO NOT high salt concentrations
Why can microaerophiles tolerate oxygen even though they can't respire?
This is because of the limited capacity of these organisms to respire or because they contain some O2-sensitive molecule such as an O2-labile enzyme
why do we need aeration to grow aerobes? hint: shaking, bubbling
This is because the O2 that is consumed by the organisms during growth is not replaced fast enough by diffusion from the air. Therefore, forced aeration of liquid cultures is needed and can be achieved by either vigorously shaking the flask or tube on a shaker or by bubbling sterilized air into the medium through a fine glass tube or porous glass disc.
osmotolerant
able to grow over wide ranges of water activity or osmotic concentration
organisms designated as nonhalotolerant, halotolerant, halophilic, or extremely halophilic are to some extent a reflection of their genetic capacity to produce or _______________________________________.
accumulate compatible solutes
For the growth of aerobes, it is necessary to provide extensive ________
aeration
How do microbes reduce osmotic concentration of cytoplasm in hypotonic (example: swelling) solutions? Hypertonic conditions?
all of the above
Toxic by-products of oxygen that are known as ROSS (reactive oxygen species)
all of the above
what other factors catalyze the by products?
all of the above
Microaerophiles
are aerobes that use oxygen only when it is present at levels reduced from that in air (microoxic conditions). Meaning, can use oxygen only when it is present at levels reduced from that in air due to limited respiration or oxygen sensitivity (8-10%)
What do compatible solutes contain
are highly water-soluble organic molecules and include sugars, alcohols, and amino acid derivatives
solutes ______ water, making it less available to organisms. Hence, for organisms to thrive in high-solute environments, physiological adjustments are necessary.
bind
how is superoxide anion destroyed?
by enzyme superoxide dismutase
When an organism is transferred from a medium of high aw to one of low aw, how does one maintain positive water?
by increasing its internal solute concentration. This is possible either by pumping solutes into the cell from the environment or by synthesizing a cytoplasmic solute. In either case, the solute must not inhibit biochemical processes in the cell and is thus called a compatible solute.
aerotolerant anaerobes
can tolerate oxygen an grow in its presence even though they cannot respire
In the case of other microbes where they can't take in oxygen, what happens to them?
can't grow in the presence of oxygen and may even be killed by it
anaerobes
cannot respire oxygen and may even be killed by exposure
thioglocate broth
complex medium containing a small amount of agar, making the medium viscous but still fluid. After thioglycolate reacts with O2 throughout the tube, O2 can penetrate only near the top of the tube where the medium contacts air. Obligate aerobes grow only at the top of such tubes. Facultative organisms grow throughout the tube but grow best near the top.
compatible solutes
compounds used by cell to counteract low water activity in surrounding environment. used by a cell to maintain water balance a small molecule having osmotic properties and act as osmolytes (low molecular weight organic compounds) that help the cell to balance external osmotic pressure and cell volume.
the ______________ of compatible solute in a cell is a function of the levels of solute in its environment
concentration
catalase and peroxidase
convert H2O2 to O2 and H2O
Superoxide dismutase (SOD)
converts O2- to H2O2 and O2
what are hypersaline environments? If an organism grows in this type of environment, at what percent do these organism grow best? Note that some of them can grow even more than the standard %
environments that are more salty than seawater (3-12% NaCl)
The concentration of compatible solute in a cell is a function of the levels of solute in its environment, and adjustments are made in response to the challenge from ______________
external solutes
when a cell is in an environment with a higher external solute concentration, water will
flow out
obligate anaerobes
inhibit or killed by oxygen carry out fermentation or anaerobic respiration and cannot survive in the presence of O2
Why is the aw at such a low number (0.61)?
likely set by the physiochemical constraints on obtaining water in osmotic environments of aw less than 0.6 that cannot be overcome through biochemical adaptations by the cell.
High solute =
low aw
changes in osmotic concentrations in the environment may affect microbial cells. What is hypotonic solution?
lower osmotic concentration water enters the cell = cell swelling (and possible bursting)
The more stuff you have that are mixed with water, the value __________. Example: milk
lowers
How does an obligate aerobe differ from a facultative aerobe?
obligate anaerobe cannot survive in the presence of oxygen while facultative anaerobe can survive in the presence of oxygen.
Halotolerant
organisms can tolerate some level of dissolved solutes but grow best in the absence of the added solute. Meaning, organisms that can tolerate some additional dissolved solutes but generally grow best in the absence of the added solutes.
For the culture of anaerobes, the problem is not to __________ O2 but to _______ it
provide exclude
How do compatible solutes work? ****
pumping solutes from environment into cell synthesizing cytoplasmic solutes highly water soluble (because of what it contains)
What is the aw of pure water? What is the lower limit of aw for life?
pure water: 1 lower limit: 0.61
Aerobes
require oxygen; can grow at full oxygen tensions (air is 21% O2); respire oxygen in their metabolism
what redox indicator dye is present in thioglycolate broth so that it can signal oxic regions?
resazurin (pink when oxidized/ colorless when reduced)
what else does seawater contain?
small amounts of many other minerals and elements
water is the _______________ and its availability is an important factor affecting the growth of microorganisms
solvent of life
example of obligate anaerobes
some bacteria, archaea, few fungi, and few protozoa
What other things are included in compatible solutes?
sugars such as sucrose and trehalose, dimethylsulfoniopropionate (produced by marine algae), and glycerol, a common solute in xerophilic fungi, organisms that grow at the lowest water activities known
all strict anaerobic microorganisms lack (or have few quantities of answer 1 answer 2 these microbes can't tolerate oxygen and must grow without it
superoxide dismutase catalase
How does superoxide dismutase or superoxide reductase protect a cell?
superoxide dismutase is an enzyme that catalyzes superoxide anions by generating H2O2 and O2 from two molecules of O2. Its an essential enzyme for aerobes because it protects the cell from oxidation of lipids, proteins and DNA
