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TRANSCRIPT
Microbial Nutrition
and Growth
Chapter 6
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Microbial Nutrition
•Bacteria require a constant influx of certain substances
from their habitat
•All organisms require a source of elements such as
carbon, hydrogen, oxygen, phosphorous, potassium,
nitrogen, sulfur, calcium, iron, sodium, chloride, nitrogen, sulfur, calcium, iron, sodium, chloride,
magnesium, and certain other elements
•Essential nutrient: any substance that must be
provided to an organism
Microbial Nutrition (cont’d)
•Macronutrients: required in relatively large quantities
and play principal roles in cell structure and metabolism
- carbon, hydrogen, and oxygen
•Micronutrients: also known as trace elements
- present in much smaller amounts and are - present in much smaller amounts and are
involved in enzyme function and maintenance of
protein structure
- manganese, zinc, nickel
Microbial Nutrition (cont’d)
•Inorganic nutrient
- an atom or simple molecule that contains a
combination of atoms other than carbon and
hydrogen
- found in the crust of the earth, bodies of
water, and the atmosphere
•Organic nutrients
- contain carbon and hydrogen atoms and are
the products of living things
- simple organic molecules such as methane
- large polymers (carbohydrates, lipids,
proteins, nucleic acids)
Chemical Analysis of the Microbial Cytoplasm
•Water – 70% of all components
•Proteins
•Organic compounds – 97% of dry cell weight
•Elements CHONPS – 96% of dry cell weight•Elements CHONPS – 96% of dry cell weight
•Most chemical elements available to the cell as
compounds and not as pure elements
•Only a few types of nutrients needed to synthesize over
5,000 different compounds
Autotrophs and Their Energy Sources
•Photoautotrophs
- Photosynthetic; capture energy from light rays
and transform it to chemical energy
- produce organic molecules using CO2 that can be
used by themselves and by heterotrophs
Autotrophs and Their Energy Sources (cont’d)
•Chemoautotrophs
- chemoorganic autotrophs: use organic
compounds for energy and inorganic compounds
as a carbon source
- lithoautotrophs: rely totally on inorganic
minerals and require neither sunlight nor
organic nutrients
Heterotrophs and Their Energy Sources
•Chemoheterotrophs
- derive both carbon and energy from organic
compounds
- process these molecules through respiration
or fermentation
•Saprobes
- free living organisms that feed on organic
detritus from dead organisms
- decomposers of plant litter, animal matter, and
dead microbes
- recycle organic nutrients
Heterotrophs and Their Energy Sources (cont’d)
•Parasites
- derive nutrients from the cells or tissues of a
living host
- pathogens: cause damage to tissues or even
death
- range from viruses to helminths- range from viruses to helminths
- ectoparasites: live on the body
- endoparasites: live in the organs and tissues
Heterotrophs and Their Energy Sources (cont’d)
•Parasites (cont’d)
- intracellular parasites: live within cells such as
the leprosy bacillus and the syphilis spirochete
- obligate parasites: unable to grow outside of a
living host
- less strict parasites can be cultured artificially - less strict parasites can be cultured artificially
if provided with the correct nutrients and
environmental conditions
•The vast majority of microbes causing human disease
are chemoheterotrophs
Essential Nutrients
•Chemicals that are necessary for particular organisms, which they cannot
manufacture by themselves
•Carbon, hydrogen, oxygen, phosphate, and sulfur (CHONPS)
Other Important Nutrients
•Sodium (Na): important for certain types of cell transport
•Calcium (Ca): stabilizer of cell wall and endospores of
bacteria
•Magnesium (Mg): component of chlorophyll and a
stabilizer of membranes and ribosomes
•Iron (Fe): important component of the cytochrome
proteins of cell respiration
Other Important Nutrients (cont’d)
•Zinc (Zn): essential regulatory element for eukaryotic
genetics
- major component of “zinc fingers;” binding
factors that help enzymes adhere to specific sites
on DNA
•Copper, cobalt, nickel, molybdenum, manganese, •Copper, cobalt, nickel, molybdenum, manganese,
silicon, iodine, and boron are needed in small amounts
by some microbes, but not others
•Metals can be toxic to microbes
•The concentration of metal ions can influence the
diseases microbes cause
How Microbes Eat:
Transport Mechanisms
•Transport of necessary nutrients occurs across the cell
membrane, even in organisms with cell walls
•The driving force of transport is atomic and molecular
movementmovement
•Diffusion: the phenomenon of molecular movement,
in which atoms or molecules move in a gradient from an
area of higher density or concentration to an area of
lower density or concentration
Diffusion
•All molecules (solid, liquid, or gas) are in continuous
movement
•As temperature increases, molecular movement
becomes faster
•In any solution, including cytoplasm, these moving
molecules cannot travel very far without having
collisions with other molecules
•As a result of these collisions, the directions of colliding
molecules are altered and unpredictable
Diffusion (cont’d)
•If the solute is more concentrated in one area than
another, the thermal movement will eventually
distribute the molecules evenly
•Diffusion of molecules across the cell membrane is
largely determined by the concentration gradient and
permeability of the substance
The Movement of Water: Osmosis
•Osmosis: the diffusion of water through a selectively, or
differentially, permeable membrane
- has passageways that allow free diffusion of water,
but block certain other dissolved molecules
- when the membrane is placed between solutions
of differing concentrations of solute and the solute
cannot pass through the membrane, water will cannot pass through the membrane, water will
diffuse at a faster rate from the side that has more
water to the side that has less water
- this will continue until the concentration of water
is equalized on both sides of the membrane
Model System to Demonstrate Osmosis
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As the H 2O diffuses into thesac, the volume increasesand forces the excesssolution into the tube, whichwill rise continually.
Even as the solution outside the sacbecomes diluted, there will still beosmosis into the sac. Equilibrium willnot occur because the solutions cannever become equal. (Why?)
2 3
Solute
Water
Glasstube
Inset shows a close-up of the osmotic process. Thegradient goes from the outer container (higherconcentration of H 2O) to the sac (lower concentration ofH2O). Some water will diffuse in the opposite directi onbut the net gradient favors osmosis into the sac.
1
Membrane sacwith solution
Containerwith water
Pore
Osmosis (cont’d)
•Living membranes generally block the entrance and exit
of larger molecules and permit the free movement of
water
•Most cells are surrounded by some free water and the
amount of water entering or leaving has a major impact
on cellular activities and survival
•This osmotic relationship between cells and their
environment is determined by the relative concentrations
of the solutions on either side of the cell membrane
Cell Responses to Solutions of Differing Osmotic Content
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Water concentration is equal insideand outside the cell, thus rates ofdiffusion are equal in both directions.
Net diffusion of water is into the cell; thisswells the protoplast and pushes it tightlyagainst the wall; wall usually preventscell from bursting.
Water diffuses out of the cell andshrinks the cell membrane away fromthe cell wall; process is known asplasmolysis.
Cel
ls w
ith c
ell w
all
Cell membrane
Isotonic solution
Osmosis
Hypotonic solution Hypertonic solution
Cell wall
Cell membrane
Cel
ls w
ithou
t cel
l wal
l
cell from bursting. plasmolysis.
Rates of diffusion are equal inboth directions.
Diffusion of water into the cell causesit to swell, and may burst it if nomechanism exists to remove the water.
Water diffusing out of the cell causesit to shrink and become distorted.
Solute
Net water movement
Osmosis (cont’d)
•Cell membranes and cell walls hinder simple diffusion
by adding a physical barrier
•Simple diffusion is limited to small nonpolar molecules
such as oxygen or lipid soluble molecules that may pass such as oxygen or lipid soluble molecules that may pass
through membranes
•It is imperative that cells move polar molecules and
ions across the plasma membrane
Transport
•The process of moving molecules into or out of cells
•Features of active transport
- the transport of nutrients against the diffusion
gradient or in the same direction as the natural
gradient, but at a rate faster than by diffusion alone
- the presence of specific membrane proteins - the presence of specific membrane proteins
(permeases and pumps)
- the expenditure of energy
•Examples of substances transported actively are
monosaccharides, amino acids, organic acids, phosphates,
and metal ions
Endocytosis:
Eating and Drinking by Cells
•Some eukaryotic cells transport large molecules,
particles, liquids, or other cells across the cell
membrane requiring the expenditure of energy
•Endocytosis
- cell encloses the substance in its membrane
- simultaneously forms a vacuole and engulfs the
substance
Endocytosis:
Eating and Drinking by Cells (cont’d)
•Phagocytosis
- accomplished by amoebas and white blood cells
- ingest whole cells or large solid matter
•Pinocytosis: ingestion of liquids such as oils or
molecules in solution
Transport Processes in Cells
Simplediffusion
A fundamentalproperty of atoms andmolecules that existin a state of randommotion
None. Substancesmove ona gradientfrom higherconcentrationto lowerconcentration.
Facilitateddiffusion
Molecule binds to aspecific receptor inmembrane and iscarried to other side.Molecule-specific. Goesboth directions. Rate oftransport is limited bythe number of bindingsites on transportproteins.
None. Substancesmove ona gradientfrom higherconcentrationto lowerconcentration.
Carrier-mediated
Atoms or molecules arepumped into or out of
Driven by ATP orthe proton motive
Table 6.4 Transport Processes in Cells
Examples DescriptionEnergyRequirements
Passive
Active
Protein
Intracellular
Membrane
ExtracellularIntracellularExtracellular
Membrane
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mediatedactivetransport
pumped into or out ofthe cell by specializedreceptors.
the proton motiveforce
Grouptranslocation
Molecule is movedacross membraneand simultaneouslyconverted to ametabolically usefulsubstance
Bulk transport Mass transport of largeparticles, cells, andliquids by engulfmentand vesicle formation.Processes generallycalled endocytosis.Phagocytosis movessolids into cell;pinocytosis movesliquids into cell.
Liquid enclosedby microvilli
Vesiclewith liquid
ATP
ATP
Vacuoles
ATPATP
Oildroplet
MicrovilliPseudopods
PinocytosisPhagocytosis
ATP
Protein
IntracellularExtracellular
Membrane
IntracellularExtracellular
Protein
ATP
Environmental Factors That Influence Microbes
•Microbes are exposed to a wide variety of factors in
addition to nutrients
•Environmental factors affect the function of metabolic
enzymesenzymes
•Survival in a changing environment is largely a matter of
whether the enzyme systems of microorganisms can
adapt to alterations in their habitat
Ecological Groups by Temperature of Adaptation
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Rat
e of
Gro
wth
PsychrophilePsychrotrophMesophileThermophileExtreme thermophile
Optimum
MinimumMaximum
0
Rat
e of
Gro
wth
Temperature °C
-20 -10 10 20 30 40 50 60 70 80 90 100 110 120 130
Environmental Factor: Gases
•The atmospheric gases that influence microbial growth
are O2 and CO2
- O2 has the greatest impact on microbial growth
- O2 is an important respiratory gas and a powerful
oxidizing agent
•Microbes fall into one of three categories•Microbes fall into one of three categories
- those that use oxygen and detoxify it
- those that can neither use oxygen nor detoxify it
- those that do not use oxygen but can detoxify it
How Microbes Process Oxygen
•As oxygen enters cellular reactions, it is transformed into
several toxic products
- singlet oxygen (O): an extremely reactive
molecule that can damage and destroy a cell by
the oxidation of membrane lipids
- superoxide ion (O2-): highly reactive
- hydrogen peroxide (H2O2): toxic to cells and
used as a disinfectant
- hydroxyl radicals (OH-): also highly reactive
How Microbes Process Oxygen (cont’d)
•Most cells have developed enzymes that scavenge and
neutralize reactive oxygen byproducts
•Two-step process requires two enzymes
• Superoxide ion is converted into hydrogen peroxide
by superoxide dismutase
• Hydrogen peroxide is converted into harmless water
and oxygen by catalase
Carbon Dioxide
•Capnophiles: organisms that grow best at a higher CO2
tension than is normally present in the atmosphere
•Important in the initial isolation of the following
organisms from clinical specimens
- Neisseria (gonorrhea, meningitis)- Neisseria (gonorrhea, meningitis)
- Brucella (undulant fever)
- Streptococcus pneumoniae
Environmental Factor: Osmotic Pressure
•Osmophiles: live in habitats with high solute
concentration
•Halophiles: prefer high concentration of salt
- Obligate halophiles Halobacterium and
Halococcus grow optimally at solutions of 25%
NaCl but require at least 9% NaClNaCl but require at least 9% NaCl
- Facultative halophiles: remarkably resistant to
salt, even though they do not normally reside in
high salt environments
- Staphylococcus aureus can grow on NaCl
media ranging from 0.1% to 20%
Associations Between Organisms
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Associations Between Organisms
Organisms live in closenutritional relationships;
required by one or both members.
Organisms are free-living;relationships not required
for survival.
Symbiotic Non symbiotic
required by one or both members.
MutualismObligatory,dependent;
both membersbenefit.
CommensalismThe commensalbenefits; other
member notharmed.
ParasitismParasite isdependent
and benefits;host harmed.
SynergismMemberscooperateand sharenutrients.
AntagonismSome members
are inhibitedor destroyed
by others.
for survival.
Strong Partnerships: Symbioses
•Symbiosis: a general term to denote a situation in which
two organisms live together in a close partnership
- symbionts: members of a symbiosis
•Three main types of symbiosis occur
- Mutualism: organisms live in an obligatory but
mutually beneficial relationship
- Commensalism: the partner called the commensal
receives benefits, while its partner is neither
harmed nor benefitted
- Parasitism: a relationship in which the host
organism provides the parasitic microbe with
nutrients and a habitat; parasite usually harms
the host to some extent
Associations but Not Partnerships:
Antagonism and Synergism
•Antagonism: an association between free-living species
that arises when members of a community compete
•Synergism:
- an interrelationship
between two organisms that between two organisms that
benefits them but is not
necessary for survival
Biofilms: The Epitome of Synergy
•Biofilms
- mixed communities of bacteria and other
microbes that are attached to a surface and each microbes that are attached to a surface and each
other
- form a multilayer conglomerate of cells and
intracellular material
Steps in the Formation of a Biofilm
Pioneer bacteria colonizea surface.
1
Pioneers secreteextracellular material thathelps keep them on thesurface and serves asattachment point for latercolonizers. Quorumsensing chemicals(red dots) are released bybacteria.
2
In many (but not all)biofilms, other speciesjoin and may contributeto the extracellular matrixand/or participate inquorum sensing with theirown chemicals or the
3
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
own chemicals or theones released by otherspecies.
Biofilms serve asa constant source ofbacteria that can “escape”and become free-livingagain.
4
Bacteria
Gauze fiber
Extracellularmatrix
Courtesy of Ellen Swogger and Garth James, Center for BiofilmEngineering,Montana State University
Biofilms: The Epitome of Synergy (cont’d)
•Quorum sensing: used by bacteria to interact with
members of the same species as well as members of
other species that are close by
•Structure of the biofilm
- large, complex communities form with different
physical and biological characteristics
- the bottom may have very different pH and
oxygen conditions than the surface
- partnership among multiple microbial
inhabitants
- cannot be eradicated by traditional methods
The Study of Bacterial Growth
• Binary fission
- one cell becomes two
- parent cell enlarges
- duplicates its chromosome- duplicates its chromosome
- starts to pull its cell envelope together to the
center of the cell
- cell wall eventually forms a complete central
septum
The Population Growth Curve
•In closed systems or batch cultures, numerous factors prevent
cells from continuously dividing at their maximum rate
•Growth curve: a predicable pattern of a bacterial population
growth in a closed system can be measured by
Crecimiento Poblacional BacterianoEl ciclo de la curva de crecimiento
Fase Lag : periodo de aclimatacióna condiciones de crecimiento, síntesisde RNA, duplicación DNA
Fase Exponencial : número de células se duplica a intervalos regulares de tiempo, ocurre bajo condiciones idealesde crecimiento (Ej. .abundancia de nutrientes)
Fase Estacionaria : se agotan nutrientes,se acumulan desperdicios, procesos dedivisión celular y muerte están en balance
Fase de Muerte : las condicionesprevalecientes no pueden sostener más crecimiento y las células mueren
Crecimiento Poblacional Bacteriano
crecimiento exponencial : número de células se duplica a intervalos regulares de tiempo
datos de una población que se duplica cada 30 minutos
Analyzing Population Size Without Culturing
•Turbidity/turbidometry
- a clear nutrient solution becomes turbid or
cloudy as microbes grow in it
- the greater the turbidity, the larger the
population size
•Counting•Counting
- direct cell count: measured microscopically
- Coulter counter: electronically scans a fluid as it
passes through a tiny pipette
- flow cytometer: works similarly to a Coulter
counter, but can measure cell size and
differentiate between live and dead cells
Medidas de Crecimiento MicrobianoMedidas de Crecimiento Microbiano
Enumeración Indirecta : turbidez/espectrofotómetro
incidentlight
detected600nm
detectedlight
absorbancia a 600nmaumenta según aumenta el# de células
se miden lecturas de absorbancia a 600nm
Steps in a Viable Plate CountCopyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
600 min
500 mlinoculatedflask
Sample is dilutedin liquid agarmedium andpoured or spreadover surface of
60 min 120 min 180 min 240 min 300 min 360 min 420 min 480 min 540 min
0.1 ml sample added to tube
Equally spacedtime intervals
2 4 7
over surface ofsolidified medium
Plates areincubated,colonies arecounted
Number ofcolonies (CFU)per 0.1 ml
Total estimatedcell populationin flask
*Only means that too few cells are present to be as sayed.
<5,000 10,000 20,000 35,000 65,000 115,000 225,000 400,000 675,000 1,150,000
23013580452313<1*
None
The Rate of Population Growth
•Generation time or doubling time: the time required
for a complete fission cycle, from parent cell to two
daughter cells
- generation: increases the population by a factor
of two
- as long as the environment remains favorable,
the doubling effect can continue at a constant
rate
The Rate of Population Growth (cont’d)
•The length of the generation time is a measure of the
growth rate of an organism
- average generation time is 30 – 60 minutes
- shortest generation times can be 10 – 12
minutes
- Mycobacterium leprae has a generation time of
10 – 30 days
- environmental bacteria have generation times
measured in months
- most pathogens have relatively short generation
times
turbidez semide a
Medidas de Crecimiento MicrobianoMedidas de Crecimiento Microbiano
Enumeración Indirecta : turbidez/espectrofotómetro
log
Abs
orba
nce
550-
600n
m
0.2
0.3
0.4
0.5
0.6
0.7
log
bact
eria
l num
bers
/ml
se determina el número de células asociado acada lectura de absorbancia(curva estándar)
mide a intervalos regulares
minutes
30 60 90 120 150 180 210 240
log
Abs
orba
nce
550
0.1
0.2
log
bact
eria
l num
bers
/ml
Direct count
g (tiempo de generación):= t(Af)-t(Ai)
90-60=30 min
Matemática de crecimiento microbiano(una vez se obtiene crecimiento exponencial)
k= velocidad de crecimiento,número de generaciones por
log
bact
eria
l num
bers
/ml
log
Abs
orba
nce
550-
600n
m0.3
0.4
0.5
0.6
0.7
k ==== (logNf – log No) / t
número de generaciones porunidad de tiempo
No=número de células inicial
Nf = No=número de células final
30 60 90 120 150 180 210 240
min
log
bact
eria
l num
bers
/ml
log
Abs
orba
nce
550
0.1
0.2
0.3