BACTERIAL NUTRITION AND METABOLISM

Rainelda Uy-Veloso, M.D.

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 hydrolytic reactions ( 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 form new chemical bonds = e.g. of anabolic processes: formation of proteins from amino acids nucleic acids from nucleotides polysaccharides from simple sugars

 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 resynthesize ATP ADP + P + energy ATP Anabolic reactions are coupled to ATP breakdown and catabolic reactions are coupled to ATP 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

 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 ways e.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 substrates and each catalyzes only one reaction

 Names usually end in ase

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 portion Apoenzyme 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

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) reactions 2. 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)

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 denaturation loss 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

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)

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

ENERGY PRODUCTION

1. Oxidation reduction
2. ATP generation Oxidation Reduction ( 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

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.g. Obligate anerobes

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

 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 two three - 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

 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 lactic acid ( homolactic or homofermentative) Can result in food spoilage but can also produce yogurt from milk and pickles from cucumbers

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 as Heterolactic 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

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 positive reaction 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

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 to convert CO2 from atmosphere to more reduced carbon compounds primarily sugars Photo means light and synthesis refers 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 oxidation reduction reactions of inorganic or organic compounds for energy

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

MICROBIAL GROWTH

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

microbes that are growing are increasing in number, accumulating into clumps of 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

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

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 in growth medium e.g. buffers: peptones, amino acids, phosphate salts

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 away from the cell wall

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

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

 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 of media 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

 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 for energy-yielding reactions

= e.g. genus Clostridium

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 oxygen 3. Must be sterile must 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 = slants solidified with the tube held at an angle In petri plates = shallow dishes with a lid that nests over the bottom to prevent contamination

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

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

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 that is 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 will discourage 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 of gram (+) bacteria

Types of Culture Media: __________________________________________________________________ Type of Media Purpose __________________________________________________________________

Chemically defined
Complex Reducing Selective Differential Enrichment

Grow chemoautotrophs and photoautotrophs and for microbial assays

Grow most chemoheterotrophic organisms Grow obligate anaerobes Suppress unwanted microbes, encourage desired microbes Distinguish colonies of desired microbes from others Similar to selective media but designed to increase numbers of desired microbes to detectable levels

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

PRESERVING BACTERIAL CULTURES Two most common methods: 1. deep freezing 2. 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

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

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

Binary Fission in Bacteria

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 phase 4. Death phase

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

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 the previous phase or the population might die out entirely

Bacterial Growth Curve

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.

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 too small 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 slide Petroff-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

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 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

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: 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 & has the 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: 3 phases of spore germination: 1. activation stage = conditions the spores to germinate in a suitable environment 2. germination stage = the characteristic properties of the dormant spore 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