2. Dr. Veloso Bacterial Nutrition and Metabolism

8/22/2019 2. Dr. Veloso Bacterial Nutrition and Metabolism

1/47

BACTERIAL NUTRITION AND METABOLISM

Rainelda Uy-Veloso, M.D.


2/47

MICROBIAL METABOLISM

Catabolic and Anabolic Reactions:

Metabolism = sum of all chemical reactions within a living organism

= 2 classes of chemical reactions:

1. those that release energy = Catabolism

2. those that require energy = Anabolism

Catabolism = breakdown of complex organic compounds into simpler ones

= reactions involved is called catabolic or degradative reactions hydrolyticreactions ( use water and in which chemical bonds are broken) chemical

bonds store energy broken chemical energy released

Anabolism = building of complex organic molecules from simpler ones

= reactions involved is called anabolic or biosynthetic reactions dehydration

synthesis reactions ( reactions that release water) require energy to formnew chemical bonds

= e.g. of anabolic processes: formation of proteins from amino acids

nucleic acids from nucleotides

polysaccharides from simple sugars


3/47

The coupling of energy-requiring and energy-releasing reactions is made possible through

Adenosine triphosphate (ATP)

ATP = stores energy from catabolic reactions and perform cellular work

= 1 molecule consist of adenine, ribose and 3 phosphate groups

= when the terminal phosphate group splits adenosine diphosphate (ADP) formed

energy released to drive anabolic reactions

ATP ADP + P + energy

= the energy from catabolic reactions is used to combine ADP and P to resynthesizeATP

ADP + P + energy ATP

Anabolic reactions are coupled to ATP breakdown and catabolic reactions are coupled toATP synthesis

The balance flow of chemicals and energy maintains the life of a cell

Only part of the energy released from catabolism is available for cellular functions, part of

the energy is lost to the environment as heat


4/47

Cells must use energy to maintain life, it has a continuous need for new external sources

of energy

Physiologic temperature and pressure of organisms are too low for chemical reactions to

occur and maintaining the life of the organism

ENZYMES

class of proteins

can speed up chemical reactions in several wayse.g. enzyme bring two reactant molecules close together and orient them to react

lowers activation energy for the reaction without increasing the

temperature or pressure inside the cell

serve as biological catalyst = substances that can speed up a chemical reaction without

being altered themselves as catalysts, enzymes are specific = each act on specific substance called substratesand each

catalyzes only one reaction


5/47

Names usually end inase

grouped into 6 according to the type of chemical reaction they catalyze and to the specific

types of reactions they assist

e.g. oxidoreductases = involved with oxidation-reduction reactions

dehydrogenases = remove hydrogen

oxidases = add oxygen

Components of Enzymes:

consist of both protein portionApoenzyme and nonprotein component the cofactor

Apoenzyme + cofactor = Holoenzyme or whole enzyme

if cofactor is removed, the apoenzyme will not function

Cofactor can be a metal ion or complex organic molecule called Coenzyme

Metal ions: iron, copper, magnesium, manganese, zinc, calcium, cobalt


6/47

Coenzymes = assist the enzymes by accepting atoms removed from the substrate or

donating atoms required by the substrate

= some act as electron carriers, removing electrons from the substrate and

donating to other molecules

= are derived from vitamins

= important coenzymes:

1. Nicotinamide adenine dinucleotide (NAD+) = involved in catabolic

(energy-yielding) reactions2. Nicotinamide adenine dinucleotide phosphate (NADP+) = involved in

anabolic (energy-requiring) reactions

3. Coenzyme A ( CoA) = a derivative of panthotenic acid

= plays a role in the synthesis and breakdown of

fats and a series of oxidizing reactions

(Krebs cycle)


7/47

Factors Influencing Enzymatic Activity:

1. Temperature

rate of chemical reactions increases as the temperature increases

molecules move more slowly at lower temperatures than higher temp. so may not have

enough energy to cause chemical reaction

Elevation of a certain temp. = reduces rate of reaction due to denaturationloss of

characteristic three-dimensional structure breakage of hydrogen bonds and other

noncovalent bonds Denaturation substances: concentrated acids, bases, heavy metal ions ( lead, arsenic,

mercury), alcohol, and ultraviolet radiation

2. pH

most enzymes have optimum pH

When H+ concentration in the medium is changed enzymes amino acids are

affected cause denaturation


8/47

3. Substrate Concentration

There is a maximum rate at which a certain amount of enzyme can catalyze a specific

reaction

Extremely high concentration of substrates = maximum rate can be attained

4. Inhibitors

An effective way to control growth of bacteria is to control their enzymes

Certain poisons combined with enzymes prevent bacteria from functioning and die

e.g. cyanide, arsenic, mercury

2 classifications of Enzyme inhibitors:

1. Competitive inhibitors = fill the active site of an enzyme and compete with the

normal substrate for active site

= the shape and chemical structure are similar to those of

the normal substrate

= does not undergo any reaction to form products

= can be reversible or irreversible

= e.g. Sulfanilamide ( sulfa drug) inhibits the enyme

whose normal substrate is para-amino-benzoic acid(PABA)


9/47

2. Non-competetive inhibitors = do not compete with the substrate for the enzymes

active site

= binds to a site on the enzyme other than the

substrates binding site (Allosteric inhibition)

= binding causes the active site to change its shape

making it non-functional which reduces enzyme

activity

= e.g. cyanide, fluoride ( enzyme poisons)

Feedback Inhibition:

Control mechanism that stops the cell from wasting chemical resources by making more of a

substance than it needs

Generally acts on the first enzyme in a metabolic pathway, enzyme is inhibited and not

synthesized

The entire pathway shuts down and no new end-product is formed


10/47

ENERGY PRODUCTION

1. Oxidationreduction

2. ATP generation

OxidationReduction ( Redox Reaction )

Oxidation = removal of electrons from an atom or molecule

= a reaction that produces energy

Reduction = gaining of one or more electrons

Oxidation and reduction are always coupled = each time one substance is oxidized, another

is reduced

In cellular oxidations, usually involve the loss of hydrogen atoms (dehydrogenation)

Cells use them in catabolism to extract energy from nutrient molecules


11/47

BIOCHEMICAL PATHWAYS OF ENERGY PRODUCTION

Carbohydrate Catabolism

Most microorganisms oxidize carbohydrates as primary source of cellular energy

The breakdown of carbohydrate molecules to produce energy Glucose is the most common carbohydrate energy source used by cells

To produce energy from glucose, microorganisms use two general processes:

A. Respiration = complete chemical and physical process in which oxygen is

delivered to tissues or cells of the body and CO2 and H2O are

given off= an ATP generating process in which molecules are oxidized

and the final electron acceptor is almost always an inorganic

molecule

= essential feature is the electron transport chain

1. Aerobic respiration = final electron acceptor is O2

e.g. obligate aerobes

facultative anaerobes

2. Anaerobic respiration = final electron acceptor is an

inorganic molecule other than

O2 ( Nitrate, sulfate and

carbonate)e. . Obli ate anerobes


12/47

B. Fermentation = a biochemical process by which microorganism breaks

down a substance into simpler ones, esp. the creation of

alcohol from the action of yeast on sugar

Electron Transport Chain:

Consists of a sequence of carrier molecules that are capable of oxidation and reduction

In Eukaryotic cells, contained in the inner membrane of mitochondria

In Prokaryotes, found in the plasma membrane

3 classes of molecules:

1. Flavoproteins = contain flavin, a coenzyme from riboflavin ( Vitamin B2)

= capable of performing alternating oxidations and reductions

= e.g. Flavin mononucleotide (FMN)

2. Cytochromes = proteins with iron containing group ( heme)

= capable of existing alternately as reduced form (Fe2+) and

oxidized form ( Fe3+)

3. Ubiquinones ( coenzyme Q) = small non-protein carriers


13/47

Both processes usually start with the same first step, Glycolysis, but follow different

subsequent pathways

GLYCOLYSIS:

AKA Embden-Meyerhof Pathway

Splitting of sugar catalyzes the splitting of glucose, a six-carbon sugar into twothree - carbon sugars

The oxidation of glucose to pyruvic acid

FERMENTATION

Releases energy from sugars or other organic molecules: amino acids, organic acids,

purines and pyrimidines

Does not require oxygen ( but sometimes can occur in its presence)

Does not require use of the Krebs cycle or an electron transport chain Uses an inorganic molecule as the final elecron acceptor

Produces only small amounts of ATP because much of the glucose energy remains in

the chemical bonds of the organic end-products, lactic acid or ethanol


14/47

Various microorganisms can ferment various substrates; end products depend on the

particular microorganism, the substrate and the enzymes that are present and active

1. Lactic Acid Fermentation

Glycolysis is the first phase of this type of fermentation

A molecule of glucose is oxidized to two molecules of pyruvic acid

Generates the energy that is used to form the two molecules of ATP

2 important genera: Streptococcus and Lactobacillus microbes that only produce lacticacid (homolactic or

homofermentative)

Can result in food spoilage but can also produce yogurt from milk and pickles from

cucumbers


15/47

2. Alcohol Fermentation

Carried out by a number of bacteria and yeasts

The ethanol and carbon dioxide produced are waste products of yeast cells but are useful

to humans

Ethanol made by yeasts is the alcohol in alcoholic beverages

Carbon dioxide made by yeasts causes bread dough to rise

Organisms that produce lactic acid as well as other acids or alcohols are known asHeterolactic or Heterofermentative

3.Propionic Acid fermentation

major end product of fermentation by some anaerobic bacteria genus Propionibacterium, anaerobic gm(+) non-spore forming rods

acid produced by the organism from glucose or lactic acid constitutes the

characteristic taste & smell of Swiss cheese


16/47

5. Mixed Acid fermentation

most of Enterobacteriaceae

Genera Escherichia, Salmonella & Shigella

ferment sugars via pyruvate to lactic, acetic, succinic & formic acid

additional CO2, H+ & ethanol are produced

6. Butanediol fermentation

org: Enterobacter, Bacillus & Serratia

conversion of pyruvate to 2,3-butanediol is responsible for the positivereaction of methyl red reaction used in differentiation of Escherichia &

Enterobacter

7. Butyric Acid fermentation

Clostridium sp.

primary products: butyric acid, acetic acid, CO2 & Hydrogen

only obligate anaerobes form butyrate as primary fermentation products

other genera: Fusobacterium, Butyrivibrio, Eubacterium=

also produce butyric acid


17/47

Photosynthesis

a process by which organisms produce simple carbohydrates from CO2 and

hydrogen using energy or other organism cellular pigments

The conversion of light energy into chemical energy which is then used toconvert CO2 from atmosphere to more reduced carbon compounds primarily

sugars

Photo means light and synthesisrefers to assembly of organic compounds

Nutritional Patterns of Organisms

Classified metabolically according to their nutritional pattern:

- source of energy

- source of carbon

Source of Energy:

1. Phototrophs = use light as primary energy source

2. Chemothrophs = depend on oxidationreduction reactions of inorganic or

organic compounds for energy


18/47

Source of Carbon:

1. Autotrophs = self-feeders

= use carbon dioxide as source of carbon

= also referred to as lithotrophs(rock eating)

2. Heterotrophs = feeders on others

= require an organic carbon source

= also referred to as organotrophs

= where all human pathogens belong

Combination of energy and carbon sources: Photoautotrophs,

Photoheterotrophs, Chemoautotrophs, and Chemoheterotrophs


19/47

MICROBIAL GROWTH

refers to the number of cells, not the size of the cells

microbes that are growing are increasing in number, accumulating into clumpsof

hundreds, colonies( accumulations of cells large enough to be seen without a

microscope)

Requirements for Growth:

1. Physical

2. Chemical

Physical Requirements:

1. Temperature

2. pH

3. Osmotic pressure


20/47

Temperature

Most microorganisms grow well at temperatures favored by humans

3 Types based on preferred range of temperature:

1. Psychrophiles ( cold loving microbes)

organisms capable of growing at 0C but has an optimum growth

temperature at 15C

2. Mesophiles ( moderate temperature-loving microbes)

optimum growth temperature is between 25C and 40C

include most of the common spoilage and disease organisms

3. Thermophiles ( heat-loving microbes)

capable of growth at optimum temperature of 50C and 60C


21/47

Minimum growth temperature = lowest temperature at which the species will grow

Optimum growth temperature = temperature at which the species grows best

Maximum growth temperature = highest temperature at which growth is possible

The optimum temperature for many pathogenic bacteria is about 37C

pH:

Most bacteria grow best in a narrow range of pH near neutrality with

pH 6.5 and 7.5

Bacteria when cultured often produce acids that interfere with their own

growth, to neutralize the acids and maintain proper pH, buffers are added ingrowth medium

e.g. buffers: peptones, amino acids, phosphate salts


22/47

Osmotic Pressure:

Microbes obtain most of their nutrients in solution from surrounding water

They require water for growth about 80-90%

Have the effect of removing water from a cell

Microbial cells in solution that has high concentration of solutes than in the cell

(hypertonic), the cellular water passes out through the plasma membrane to

the high salt concentration shrinkage of cells plasma ( cytoplasmic)

membrane cell growth is inhibited as the plasma membrane pulls awayfrom the cell wall

Used to preserve foods by adding salts ( or other solutes) to a solution


23/47

Chemical Requirements:

1. Carbon

The structural backbone of living matter

Needed for all the organic compounds that make up a living cell

Consist of about one half of the dry weight of a typical bacterial cell

2. Nitrogen, Sulfur, and Phosphorus

Needed for the synthesis of cellular material

e.g. protein synthesis require nitrogen and sulfur

synthesis of DNA and RNA also require nitrogen and phosphorus

Nitrogen makes up about 12% to 15% of the dry weight of a bacterial cell

Sulfur and phosphorus make up about 3%

Organisms use nitrogen primarily to form the amino group of the amino

acids of proteins

Sulfur is used to synthesize sulfur-containing amino acids and vitamins such

as biotin and thiamine


24/47

Phosphorus is essential for synthesis of nucleic acids and phospholipids of cell

membranes

Potassium, magnesium, and calcium are also elements that microorganism require,

often as cofactors for enzymes

3. Trace Elements

Iron, copper, molybdenum and zinc are essential for the activity of certain

enzymes as cofactors

Usually assumed to be naturally present in tap water and other components ofmedia

4. Oxygen

Microbes that use molecular oxygen (aerobes) produce more energy from

nutrients than do microbes that do not use oxygen

Obligate aerobes = organisms that require oxygen to live

= are at a disadvantage because oxygen is poorly soluble in water

of their environment


25/47

Facultative anaerobes = organisms with the ability to continue growing in the

absence of oxygen by using fermentation and anaerobic

respiration= Efficacy in producing energy decreases in the absence of

oxygen

= e.g. E. coli and many yeasts

Obligate anaerobes = are bacteria that are unable to use molecular oxygen forenergy-yielding reactions

= e.g. genus Clostridium


26/47

CULTURE MEDIA


27/47

CULTURE MEDIA

A nutrient material prepared for the growth of microorganisms in a laboratory

The microbes that grow and multiply in or on a culture medium = Culture

Criteria of a good culture medium:

1. Must contain the right nutrients for the particular organism we want to

grow

2. It should contain sufficient moisture , properly adjusted pH and suitable

level of oxygen3. Must be sterilemust initially contain no living microorganism

4. Should be incubated at the proper temperature

Agar = a complex polysaccharide derived from a marine alga

=a solidifying agent that is used as a thickener

= usually contained in test tubes or petri plates

in test tubes = slantssolidified with the tube held at an angle

In petri plates = shallow dishes with a lid that nests over the bottom toprevent contamination


28/47

Chemically Defined Media

exact chemical composition is known

used for growth of a chemoautotrophic organism capable of extracting energy

from the oxidation of ammonium ions to nitrate ions

Some organisms require many growth factors (fastidious)

Complex Media

Media whose exact chemical composition varies slightly from batch to batch Made up of nutrients such as extracts from yeasts, meat, or plants or digests of

proteins and other sources

The energy, carbon, nitrogen and sulfur requirements are met largely by

proteins partial digestion by acids or enzymes reduces protein to shorter

chains of amino acids (peptones) can be digested by bacteria

In liquid form = nutrient broth

When agar is added = Nutrient agar

A bi G h M di d M h d


29/47

Anaerobic Growth Media and Methods

Reducing media = contain ingredients such as sodium thioglycolate that

chemically combine with dissolved oxygen and deplete the

oxygen in the culture medium

= stored in ordinary, tightly capped test tubes

= media are heated shortly before use so that any absorbed

oxygen is driven off

SELECTIVE AND DIFFERENTIAL MEDIA

detect the presence of specific microorganisms associated with disease or poor

sanitation

Selective media = designed to suppress the growth of unwanted bacteria and

encourage the growth of the desired microbes

e.g. Bismuth sulfite agar = medium used to isolate typhoid

bacterium

Sabourauds dextrose agar = used to isolate fungi

Brilliant Green agar = has a dye brilliant green that inhibits

gram (+) bacteria and used to isolate gram (-) Salmonella


30/47

Differential Media

Make it easier to distinguish colonies of the desired organism from other colonies

growing on the same plate

e.g. Blood Agar ( contains red blood cells) = reddish-brown medium thatis used to identify Streptoccocus pyogenes ( show clear

ring around the colonies

Mannitol Salt Agar medium = both selective and differential media

= contains 7.5% Sodium chloride which willdiscourage the growth of competing organisms and

select growth of S. aureus

Mc Conkey Agar = both selective and differential

= contains bile salts and crystal violet which inhibit growth ofgram (+) bacteria


31/47

Types of Culture Media:

__________________________________________________________________

Type of Media Purpose__________________________________________________________________

Chemically defined Grow chemoautotrophs and photoautotrophs and for

microbial assays

Complex Grow most chemoheterotrophic organisms

Reducing Grow obligate anaerobes

Selective Suppress unwanted microbes, encourage desired microbes

Differential Distinguish colonies of desired microbes from others

Enrichment Similar to selective media but designed to increase

numbers of desired microbes to detectable levels


32/47

Obtaining Pure Cultures

Needed to isolate a specific specie of bacteria in most infectious materials

( pus, sputum and urine)

Streak Plate method = isolation method most commonly used

= works well when the organism to be isolated is present

in large numbers relative to the population


33/47

PRESERVING BACTERIAL CULTURES

Two most common methods:

1. deep freezing2. lyophilization ( freeze-drying)

Deep Freezing:

A process in which a pure culture of microbes is placed in a suspending liquid and

quick-frozen at temperatures ranging from -50C to -95C

The culture can usually be thawed and used up to several times


34/47

Lyophilization ( freeze-drying)

A suspension of microbes is quickly frozen at temperatures ranging from -54 to

-72C, and the water is removed by a high vacuum (sublimation)

While under vacuum, the container is sealed by a high-temperature torch

The remaining powder-like residue that contains the surviving microbes can be

stored for years

The microbes can be revived at any time by hydration with a suitable liquid

nutrient medium


35/47

GROWTH OF BACTERIAL CULTURES

Bacterial Division:

Bacterial growth refers to an increase in bacterial numbers not an increase in the size

of the individual cells

Bacteria normally reproduce by binary fission

Steps:1. Cell elongates and DNA is replicated

2. Cell wall and plasma membrane begin to divide

3. Cross wall forms completely around divided DNA

4. Cells separate


36/47

Binary Fission in Bacteria


37/47

Generation Time:

The time required for a cell to divide ( and its population to double)

Most bacteria has generation time of 1-3 hours Others require more than 24 hours per generation

PHASES OF GROWTH

Four basic phases:

1. Lag Phase

2. Log phase or exponential growth phase

3. Stationary phase4. Death phase


38/47

Lag Phase:

the number of cells changes very little because the cells do not immediately

reproduce in a new medium

the period of little or no cell division

can last for an hour or several days

Log phase or Exponential Growth Phase:

the cells begin to divide and enter a period of growth or logarithmic increase

cellular reproduction is most active

the generation time reaches a constant minimum

time when cells are most active metabolically

microorganisms are particularly sensitive to adverse conditions


39/47

Stationary Phase:

the growth rate slows

the number of microbial deaths balances the number of new cells, the population

stabilizes

period of equilibrium

the exhaustion of nutrients and accumulation of waste products and harmful

changes in pH

Death Phase:

the number of deaths soon exceeds the number of new cells formed

the population is diminished to a tiny fraction of the number of cells in theprevious phase or the population might die out entirely


40/47

Bacterial Growth Curve


41/47

Direct Measurement of Microbial Growth

Methods:

1. Measure cell numbers

2. Measure the populations total mass = directly proportional to cell numbers

Plate Counts:

Most frequently used method for the measurement of bacterial populations

Advantage: measures the number of viable cells

Disadvantage: It takes some time, usually 24 hours or more for viable colonies to

form

Only plates with 25 to 250 colonies are counted

To ensure that some colony counts will be within the range, the original

inoculum is diluted several times in a process called serial dilution.


42/47

Filtration:

A method used when the quantity of bacteria is very small

100 ml or more water are passed through a thin membrane filter whose pores are toosmall to allow bacteria to pass

The filter is then transferred to a petri dish containing a pad soaked in liquid nutrient

medium

This method is applied frequently to coliform bacteria

Direct Microscopic Count:

A measured volume of a bacterial suspension is placed inside a defined area on a

microscope slidePetroff-Hausser Counter= a specially designed slide

Advantage: No incubation time is required, they are usually reserved for applications

in which time is the primary consideration


43/47

Petroff-Hausser Cell Counter

The average number of cells within a large

square multiplied by a factor of 1,250,000 gives

the number of bacteria per ml.

Estimations of Bacterial Numbers by Indirect Methods


44/47

y

1. Turbidity

As bacteria multiply in a liquid medium, the medium becomes turbid or

cloudy with cells spectrophotometer or colorimeter

As bacterial numbers increase, less light will reach the photoelectric cell

The change of light will register on the instruments scale as % of

transmission

Turbidity estimation of bacterial numbers


45/47

2. Metabolic Activity

Assumes that the amount of a certain metabolic product ( acid or CO2) is in

direct proportion to the number of bacteria present

A dye is used (methylene blue) that changes color in the presence or absence of

oxygen

3. Dry weight

A better method to measure the growth of filamentous organisms (fungi)

DIFFERENTIATION IN BACTERIAL CELLS:


46/47

SPORULATION:

unique property of certain organisms (e.g. Bacillus & Clostridium) is their

ability to form endospores

Spore = a dormant structure capable of surviving for prolonged periods & hasthe

capacity to reestablish the vegetative stage of growth under

appropriate

environmental conditions

Endospore formation= during stationary phase of growth after the depletion of

nutrients in culture medium esp. carbon & nitrogen

source

GERMINATION


47/47

GERMINATION:

3 phases of spore germination:

1. activation stage = conditions the spores to germinate in a suitableenvironment

2. germination stage = the characteristic properties of the dormantspore are lost

3. outgrowth stage = spore is converted into new vegetative cell

spores germinate very slowly unless activated by heat or various chemical

treatments germination is an irreversible process = triggered by L-Alanine the most

common nutrient germinant

= other germinants: amino acids

nucleosides

glucose

2. Dr. Veloso Bacterial Nutrition and Metabolism

Documents

Transcript of 2. Dr. Veloso Bacterial Nutrition and Metabolism