microbial nutrition and growthacademic.uprm.edu/~lrios/3725/chapter6.pdfmicrobial nutrition...

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

Nutritional Categories of Microbes

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 of Molecules in Aqueous Solutions

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


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


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


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


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


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


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

Miremos en detalle!!

How do microorganisms grow?

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

Can we prevent the death phase? How?

microbial nutrition and growthacademic.uprm.edu/~lrios/3725/chapter6.pdfmicrobial nutrition...

Documents