MICROBIAL NUTRITION, GROWTH AND METABOLISM Unit II

MICROBIAL NUTRITION, GROWTH AND METABOLISM Unit II

MICROBIAL NUTRITION, GROWTH AND METABOLISM
– Bacteria Nutritional requirements Growth curve Different methods to quantify the growth Nutrient uptake Bioenergetics- Utilization of energy in Biosynthesis of important molecules Synthesis of macromolecule peptidoglycan Synthesis of small amino acid Synthesis of organic cell material in chemoautotrophic bacteria Metabolism

For You to Grow….

HOW ABOUT THEM?

Nutrients Nutrients are substances that provides nourishment essential for the maintenance of life and for growth. Nutrients are used in biosynthesis and energy production and therefore are required for microbial growth.

REQUIREMENTS FOR BACTERIAL GROWTH
Nutrients that play an important role in growth and development of all living organisms. The growth of bacteria are basically required TWO Nutritional Factors: Physical Requirements Chemical Requirements

REQUIREMENTS FOR BACTERIAL GROWTH
PHYSICAL REQUIREMENTS CHEMICAL REQUIREMENTS Temperature pH Oxygen Hydrostatic Pressure Osmotic pressure Macronutrients Carbon Nitrogen, sulfur, and phosphorous Micronutrients Trace elements Oxygen Organic growth factor

REQUIREMENTS FOR BACTERIA GROWTH: TEMPERATURE

Temperature According to their growth temperature range, bacteria can be classified as: Psychrophiles : grow best at 15-20oC Psychrotrophs : grow between 0°C and 20–30°C Mesophiles : grow best at 25-40oC Thermophiles : grow best at 50-60oC Hyperthermophiles: grow best at oC Typical Growth Rates and Temperature Minimum growth temperature: lowest temp which species can grow Optimum growth temperature: temp at which the species grow best Maximum growth temperature: highest temp at which grow is possible

Physical Factors Required for Bacterial Growth
Optimum pH: the pH at which the microorganism grows best (e.g. pH 7) Most bacteria grow between pH 6.5 and 7.5 Molds and yeasts grow between pH 5 and 6 According to their tolerance for acidity/alkalinity, bacteria are classified as: Acidophiles (acid-loving): grow best at pH Neutrophiles: grow best at pH 5.4 to 8.0 Alkaliphiles (base-loving): grow best at pH

Oxygen Aerobes: require oxygen to grow
Obligate aerobes: must have free oxygen for aerobic respiration (e.g. Pseudomonas) Anaerobes: do not require oxygen to grow Obligate anaerobes: killed by free oxygen (e.g. Bacteroides) Microaerophiles: grow best in presence of small amount of free oxygen Capnophiles: carbon-dioxide loving organisms that thrive under conditions of low oxygen Facultative anaerobes: carry on aerobic metabolism when oxygen is present, but shift to anaerobic metabolism when oxygen is absent Aerotolerant anaerobes: can survive in the presence of oxygen but do not use it in their metabolism Obligate: organism must have specified environmental condition Facultative: organism is able to adjust to and tolerate environmental condition, but can also live in other conditions

Hydrostatic Pressure Water in oceans and lakes exerts pressure exerted by standing water, in proportion to its depth Pressure doubles with every 10 meter increase in depth Barophiles: bacteria that live at high pressures, but die if left in laboratory at standard atmospheric pressure

Osmotic Pressure Environments that contain dissolved substances exert osmotic pressure, and pressure can exceed that exerted by dissolved substances in cells Hyperosmotic environments: cells lose water and undergo plasmolysis (shrinking of cell) Hypoosmotic environment: cells gain water and swell and burst

Plasmolysis

Halophiles Salt-loving organisms which require moderate to large quantities of salt (sodium chloride) Membrane transport systems actively transport sodium ions out of cells and concentrate potassium ions inside Why do halophiles require sodium? 1) Cells need sodium to maintain a high intracellular potassium concentration for enzymatic function 2) Cells need sodium to maintain the integrity of their cell walls

Responses to Salt

The Great Salt Lake in Utah

Chemical Requirement: Nutritional Factors
Macronutrients Micronutrients Trace elements (e.g. copper, iron, zinc, and cobalt) Vitamins (e.g. folic acid, vitamin B-12, vitamin K)

Nutritional Requirements

Nutritional Requirements
Micro Macro Trace

Essentials of Bacterial nutrition
1. Macronutrients needed in larger amounts eg:(CHONPS) Carbon, hydrogen, oxygen, nitrogen, phosphorous, and sulfur. H and O are common. Sources of C, N, P, and S must also be provided. 2. Micronutrients Micronutrients needed in smaller amounts: Mineral salts such as Ca+2, Fe+3, Mg+2, K+ 3. Trace elements; needed in very tiny amounts; e.g. Zn+2, Mo+2, Mn+2

Nutritional Element Use
Carbon Oxygen Nitrogen Hydrogen Phosphorus Sulfur Main component Cell water, aerobic respiration AA, coenzymes H2O Nucleotides, PL, LPS Several AA; coenzyme

Major nutritional type
Nutritional types of microorganisms Major nutritional type Sources of energy, hydrogen/electrons, and carbon Representative microorganisms Photoautotroph (Photolithotroph) Light energy, inorganic hydrogen/electron(H/e-) donor, CO2 carbon source Algae, Purple and green bacteria, Cyanobacteria Photoheterotroph (Photoorganotroph) Light energy, inorganic H/e- donor, Organic carbon source Purple nonsulfur bacteria, Green sulfur bacteria Chemoautotroph (Chemolithotroph) Chemical energy source (inorganic), Inorganic H/e- donor, CO2 carbon source Sulfur-oxdizing bacteria, Hydrogen bacteria, Nitrifying bacteria Chemoheterotroph (Chenoorganotroph) Chemical energy source (organic), Organic H/e- donor, Organic carbon source Most bacteria, fungi, protozoa

CO2 + H2O Light + Chlorophyll （CH2O） +O2
Photoautotroph Algae, Cyanobacteria CO2 + H2O Light + Chlorophyll （CH2O） +O2 Purple and green bacteria CO2 + 2H2S Light + bacteriochlorophyll （CH2O） + H2O + 2S Photoheterotroph Purple nonsulfur bacteria (Rhodospirillum) CO2 + 2CH3CHOHCH3 Light + bacteriochlorophyll （CH2O） + H2O + 2CH3COCH3

Properties of microbial photosynthetic systems
Property Cyanobacteria Green and purple bacteria Purple nonsulfur bacteria Photo - pigment Chlorophyll Bcteriochlorophyll O2 production Yes No Electron donors H2O H2, H2S, S Carbon source CO2 Organic / CO2 Primary products of energy conversion ATP + NADPH ATP

Chemoautotroph Bacteria Electron donor Electron acceptor Products H2
Alcaligens and Pseudomonas sp. H2 O2 H2O Nitrobacter NO2- NO3- , H2O Nitrosomonas NH4+ NO2- , H2O Desulfovibrio SO4 2- H2O. H2S Thiobacillus denitrificans S0. H2S NO3- SO4 2- , N2 Thiobacillus ferrooxidans Fe2+ Fe3+ , H2O Nitrifying bacteria 2 NH O NO H2O + 4 H Kcal

Culture media

Culture Media (an overview)
Culture Medium – a nutrient material prepared for the growth of microorganisms in a laboratory Inoculum – when microbes are introduced into a culture medium to initiate growth Sterile – initially containing no living organisms Agar – a complex polysaccharide derived from a marine alga which has long been used as a thickener in foods such as jellies and ice cream

Cont… Slants – what test tubes are called when agar is allowed to solidify with the tube held at an angle so that a large surface area for growth is available Deep – what test tubes are called when the agar solidifies vertically within the tube Petri (or culture) plates – what Petri dishes are called when filled

Petri dish/Petri plate
Julius Richard Petri 1887 invented the Petri dish Vital tool for microbes cultivation

Microbiological Media
Culture media are needed to grow microorganisms in the laboratory and to carry out specialized procedures like microbial identification, water and food analysis, and the isolation of particular microorganisms. A wide variety of media is available for these and other purposes.

Microbiological Media
Liquid medium Components are dissolved in water and without added Agar Solid medium A medium to which has been added a solidifying agent Agar (2.0%) Semisolid medium Agar (most commonly used at low percentage 0.8%) Silica gel (used when a non-organic gelling agent is required)

Agar Complex polysaccharide
Used as solidifying agent for culture media in Petri plates, slants, and deeps Generally not metabolized by microbes Liquefies at 100°C Solidifies at ~40°C Isolated from red algae

Types of Culture Media Natural Media: In nature, many species of microorganisms grow together in oceans, lakes, and soil and on living or dead organic matter Synthetic medium: A medium prepared in the laboratory from material of precise or reasonably well-defined composition Complex medium: contains reasonably familiar material but varies slightly in chemical composition from batch to batch (e.g. peptone, a product of enzyme digestion of proteins)

Selective, Differential, and Enrichment Media
Selective medium: encourages growth of some organisms but suppresses growth of others (e.g. antibiotics) Differential medium: contains a constituent that causes an observable change (e.g. MacConkey agar) Enrichment medium: contains special nutrients that allow growth of a particular organism that might not otherwise be present in sufficient numbers to allow it to be isolated and identified

Types of Culture Media Type of Media Purpose Chemically Defined
Growth of chemoheterotrophs and photoautotrophs: microbiological assays Complex Growth of most chemoheterotrophic organisms Reducing Growth of obligate anO2 Selective Suppresion of unwanted microbes; encouraging desired microbes Differential Differentiation of colonies of desired microbes from others Enrichment Similar to selective media but designed to increase numbers of desired microbes to detectable levels.

Selective Media Suppress unwanted microbes and encourage desired microbes Ex: Sabouraud’s Dextrose Agar: used to isolate fungi, has a pH of 5.6, outgrow most of bacteria

Differential Media Make it easy to distinguish colonies of different microbes. Ex: Blood Agar: bacteria that can lysed blood cells causing a clear areas around the colonies. Mac Conkey Agar: Lactose fermenting bacteria and non lactose fermenting Gram negative bacteria-sodium taurocholate –Gram negative bacteria inhibitor

Identification of urinary tract pathogens with
differential media (CHROMagar) Three species of Candida can be differentiated in mixed culture when grown on CHROMagar Candida plates

Differential Media

Enrichment Media Encourages growth of desired microbe
Assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria Inoculate phenol-containing culture medium with the soil, and incubate Transfer 1 ml to another flask of the phenol medium, and incubate Again transfer 1 ml to another flask of the phenol medium, and incubate Only phenol-metabolizing bacteria will be growing

Anaerobic Growth Media and Methods
Must use reducing media that contain chemicals like sodium thioglycolate that combine with oxygen to deplete it Labs may have special incubators for anaerobes or capnophiles (microbes that grow better with increased carbon dioxide)

Culturing Bacteria Culturing of bacteria in the laboratory presents two problems: 1. A pure culture of a single species is needed to study an organism’s characteristics 2. A medium must be found that will support growth of the desired organism

There are various streaking methods to get a pure colony.
CULTURING TECHNIQUES There are various streaking methods to get a pure colony. Simple streak: The inoculation loop is sterilized by heating it in the flame till it becomes red hot. The loop is dipped in the medium containing the organisms and a smear is made on one corner of the plate. This is called the mother smear. From this smear a simple streak is done. T- streak: Make a mother smear at one corner of the plate and make a few streaks from this smear. Continue streaking from one of the made streaks to obtain a Tstreak. Quadrant streak: Make a mother smear at one corner of the plate and make a few streaks from it. Rotate the plate and make the second set of streaks from the first set. Repeat the procedure until you get a quadrant of streaks.

Simple streak and T- streak:
Quadrant streak

Obtaining Pure Cultures
Pure culture: a culture that contains only a single species or strain of organism A colony is a population of cells arising from a single cell or spore or from a group of attached cells A colony is often called a colony-forming unit (CFU) The streak plate method is used to isolate pure cultures

Factors that Influence Growth
Temperature Most bacteria grow throughout a range of approximately 20°C degrees, with the maximum growth rate at a certain “optimum temperature” Psychrophiles: Grows well at 0ºC; optimally between 0ºC – 15ºC Psychrotrophs: Can grow at 0 – 10ºC; optimum between 20 – 30ºC and maximum around 35ºC Mesophiles: Optimum around 20 – 45ºC Moderate thermophiles: Optimum around 55 – 65 ºC Extreme thermophiles (Hyperthermophiles): Optimum around 80 – 113 ºC

pH Salt concentration Acidophiles: Neutrophiles Alkalophiles
Grow optimally between ~pH 0 and 5.5 Neutrophiles Growoptimally between pH 5.5 and 8 Alkalophiles Grow optimally between pH 8 – 11.5 Salt concentration Halophiles require elevated salt concentrations to grow; often require 0.2 M ionic strength or greater and may some may grow at 1 M or greater; example, Halobacterium Osmotolerant (halotolerant) organisms grow over a wide range of salt concentrations or ionic strengths; for example, Staphylococcus aureus

Oxygen concentration Strict aerobes: Require oxygen for growth (~20%)
Strict anaerobes: Grow in the absence of oxygen; cannot grow in the presence of oxygen Facultative anaerobes: Grow best in the presence of oxygen, but are able to grow (at reduced rates) in the absence of oxygen Aerotolerant anaerobes: Can grow equally well in the presence or absence of oxygen Microaerophiles: Require reduced concentrations of oxygen (~2 – 10%) for growth

BACTERIAL GROWTH

Bacterial growth “Growth” is generally used to refer to the acquisition of biomass leading to cell division, or reproduction Bacterial Growth - an increase in bacterial numbers - does not refer to an increase in size of the individual cells

Bacterial growth Kinetics: batch culture

Bacterial growth Kinetics: batch culture
Bacterial Growth Kinetics describe how the microbe grows in the fermenter. This information is important to determine optimal batch times. The growth of microbes in a fermenter can be broken down into four stages: Lag Phase Exponential Phase/log phase Stationary Phase Death Phase 3 4 2 1

Bacterial Growth Kinetics
Lag Phase This is the first phase in the fermentation process The cells have just been injected into a new environment and they need time to adjust accordingly Cell growth is minimal in this phase. There is no product synthesis

Exponential Phase/Log phase The second phase in the fermentation process The cells have completely adjusted to their environment and rapid growth takes place Cell growth rate is highest in this phase But the product synthesized is low

Exponential Phase (Continued) At some point the cell growth rate will level off and become constant The most likely cause of this leveling off is substrate limited inhibition Substrate limited inhibition means that the microbes do not have enough nutrients in the medium to continue multiplying.

Stationary phase This is the third phase in the fermentation process The cell growth rate has leveled off and become constant The number of cells multiplying equals the number of cells dying cryptic growth (cell growth rate =cell death rate) secondary metabolites are produced

Death phase The fourth phase in the fermentation process The number of cells dying is greater than the number of cells multiplying The cause of the death phase is usually that the cells have consumed most of the nutrients in the medium and there is not enough left for sustainability

Microbial Growth Kinetics

Bacterial growth t = generation time Growth = increase in # of cells
(by binary fission) generation time: 10 min - days Growth rate = Δcell number/time or Δcell mass/time t = generation time

Generation Time The generation time among organisms vary according to environmental conditions such as temperature or pH level. Most bacteria have a generation time of 1 – 3 hours while other species can require up to 24 hours per generation.

Yield Coefficient Yield coefficient (Y), is determined on the basis of the quantity of rate- limiting nutrient, normally the substrate converted into the microbial product. In case of biomass production, the yield coefficient relates to the quantity of biomass produced per gram of substrate utilized and is depicted by the equation x = Yx/s(S – Sr) x = biomass concentration (g/L), Yx/s= yield coefficient (g biomass/g substrate utilized), S =initial substrate concentration (g/L), Sr = residual substrate concentration (g/L) Therefore, the higher is the yield coefficient, the greater the percentage of the original substrate converted into microbial biomass.

Determination of yield coefficient is important as it will decide how productive and how cost viable is the medium used.

Measurement of Microbial Growth
CFU Direct Viable Plate Counts Serial Dilutions Pour Plate Spread Plate Membrane Filtration Counting Chamber MPN (fecal, H20, food) Indirect Turbidity Metabolic activity

Direct Methods Must perform - Serial Dilutions
Plate Count Must perform - Serial Dilutions - Pour plate or Spread Plate Method often reported as colony-forming units (CFU) Serial Dilution

Plate Counts Figure 6.17

Plate Counts After incubation, count colonies on plates that have 25–250 colonies (CFUs) Figure 6.16

2) Counting Bacteria by Membrane Filtration

3) The Most Probable Number (MPN) Method
Method to estimate number of cells Multiple tube MPN test Count positive tubes Compare with a statistical table.

4) Direct Microscopic Counts
Another way to measure bacterial growth by: 1) Petroff-Hausser counting chamber 2) Colony counting chamber In Petroff-Hausser counting chamber, bacterial suspension is introduced onto chamber with a calibrated pipette Microorganisms are counted in specific calibrated areas Number of cell per unit volume is calculated using the formula

Countable number of colonies
Counting colonies using a bacterial colony counter Countable number of colonies (30 to 300 per plate)

Indirect Methods 1) Turbidity
Practical way of monitoring bacterial growth. Measure turbidity using spectrophotometer A Spectrophotometer: This instrument can be used to measure bacterial growth by measuring the amount of light that passes through a suspension of cells

Turbidity The less light transmitted, the more bacteria in sample.

2) Metabolic Activity Assuming the amount of a certain metabolic product, acids, CO2 produced = direct proportion of no of bacteria present 3) Dry Weight Apply filtration of amount of broth culture on filter paper and dried in a dessicator Weight of dried culture = direct proportion of no of bacteria present

Microbial metabolism: Utilization of energy, nutrient uptake & biosynthesis of important molecules

Energy-utilized by the microorganisms
Energy-stored in the form of high-energy-transfer compounds (ATP) Energy-also available in the form of protonmotive force (electrochemical proton gradient) In these forms the energy is used to drive the many endergonic reactions required for the cell. electrochemical proton gradient – result in the ATP synthesis It can also used for other biological purposes without the synthesis of ATP Eg: used to generate heat rotation of bacterial flagella

Energy utilization in non-biosynthetic processes
ATP formed by the energy producing reactions of the bacterial cell- utilized in various ways Much energy – used in the biosynthesis of new cell components - energy-storage inclusion granules such as glycogen and poly-β-hydroxybutyrate Other metabolic processes – -physical and chemical integrity of the cell -transport of solute across membranes, -activity of locomotor organells (flagella)

Energy utilization-Bacterial motility:
maintenance of the physical and chemical integrity of the cell is mainly through reactions that lead to biosynthesis of macromolecules – nucleic acids and proteins that are continuously broken down and need replacement Energy utilization-Bacterial motility: Bacterial flagella filaments - have no machinery for interconverting chemical and mechanical energy Eg: flagellin – no enzymatic activity, i.e., no detectable ATPase activity (present in cilia and flagella of eucaryotic microorganisms (protozoa)) Thus, the bacterial flagella differ from much larger and more complex cilia and flagella - protozoa Therefore, the ATP is not the immediate source of energy for flagellar rotation

M and S ring in the basal body – rotary motor
Instead the flagellar motor is driven by the proton motive force (i.e., the force derived from the electric potential and the hydrogen gradient across the cytoplasmic membrane) M and S ring in the basal body – rotary motor The rod is fixed rigidly to the M ring – which rotates freely in the cytoplasmic membrane The S ring – mounted rigidly on the cell wall The inward flux of Protons drives the flagellar motor What molecular events cause the conversion of proton motive force – still unknown It is clear that in the flagellar rotation, proton movements constitute the energy currency and not ATP

Nutritional Uptake

Nutritional Uptake (Transport of nutrients by the bacteria
Various processes- by which the ions or molecules cross the cytoplasmic membrane cytoplasmic membrane-allows the passive passage of certain small molecules and actively concentrates others within the cell Nutrient molecules frequently cannot cross selectively permeable plasma membranes through passive diffusion and must be transported by one of three major mechanisms involving the use of membrane carrier proteins.

Nutrient Uptake Diffusion Active Transport Group Translocation Simple
Passive Facilitated Active Transport ATP H+ (proton motive) Group Translocation Alter molecule PTS (phosphorylate)

Simple diffusion It refers to a process whereby a substance passes through a membrane without the aid of an intermediary such as a integral membrane protein. In bacteria, a small noncharged molecules or lipid soluble molecules pass between the phospholipids to enter or leave the cell, moving from areas of high concentration to areas of low concentration (they move down their concentration gradient). Oxygen and carbon dioxide and most lipids enter and leave cells by simple diffusion

Passive diffusion: Except water and some lipid soluble molecules, few compounds can pass through the cytoplasmic membrane by simple or passive diffusion In this process solute molecules cross the membrane as a result of difference in concentration of the molecule across the membrane The difference in concentration (higher outside the membrane than inside) governs the rate of inward flow of the solute molecule With time, this concentration gradient diminishes until equilibrium is reached In passive diffusion, no substance in the membrane interacts specifically with the solute molecule

Facilitated diffusion:
Similar to passive diffusion Solute molecule flows from higher to lower conc. Differs from passive diffusion in that – it involves a specific protein carrier molecule (porter or permease) located in the cytoplasmic membrane Combines reversibly with the solute molecule (Carrier-solute complex) Carrier-solute complex moves between the outer and inner surfaces of the membrane, releasing one solute molecule on the inner surface and returning to bind a new one on the outer surface Eg: the entry of glycerol into bacterial cells

A model of facilitated diffusion
The membrane carrier can change conformation after binding an external molecule and subsequently release the molecule on the cell interior. It then returns to the outward oriented position and is ready to bind another solute molecule. Because there is no energy input, molecules will continue to enter only as long as their concentration is greater on the outside.

Group translocation: Solute is altered chemically during transport
Eg : phosphoenol pyruvate dependent sugar- phosphotransferase system Widely distributed in many bacterial genera and mediates the translocation of many sugars and sugar derivatives These solutes (sugars) enter the cell as sugar phosphates and are accumulated in the cell in phosphoenol pyruvate form Phosphotransferase system sugar uptake and phosphorylation require the participation of several soluble and membrane-bound enzymes These proteins catalyze the transfer of the phosphoryl group of phosphoenolpyruvate to the sugar molecule

The products formed are therefore sugar phosphate and pyruvate; the overall reaction requires Mg2+
Heat stable carrier protein (HPr) is activated first by transfer of a phosphate group from the high energy compound phosphoenol pyruvate (PEP) inside the cell Enzyme I PEP+HPr pyruvate+phospho-HPr Enzyme I and HPr are soluble proteins and non specific components of the process At the same time the sugar combines with enzyme II at the outer membrane surface and is transported to inner membrane surface

Enzyme II is specific for a particular sugar and is an integral component of the cytoplasmic membrane Here it combines with the phosphate group carried by the activated HPr The sugar phosphate is released by enzyme II and enters the cell Sugar+phospho-HPr sugar-phosphate+HPr (Outside cell) (inside cell)

Group translocation

Active transport: Active transport is the transport of solute molecules to higher concentrations, or against a concentration gradient, with the use of metabolic energy input. Almost all solutes, including sugars, amino acids, peptides, nucleosides and ions are taken up by cells through active transport 3 steps: Binding of a solute to a receptor site on a membrane bound carrier protein Translocation of the solute-carrier complex across the membrane Coupling of translocation to an energy yielding reaction to lower the affinity of the carrier protein for the solute at the inner membrane surface so that the carrier protein will release solute to the cell interior

In this, energy released during the flow of electrons through the ETC or the splitting of the phosphate group from ATP drives protons out of the cell This generates a difference in pH value and electric potential between the inside and the outside of the cell or across the membrane This proton gradient gives rise to a proton motive force which can be used to pump the solutes into the cell When proton reenter the cell, the energy released drives the transport mechanism in the cell membrane by including conformational change in the carrier molecule so that its affinity for the solute is decreased and the solute is released into the cell interior

Active transport

Facilitated diffusion Active transport Group translocation
Simple comparison of transport systems Items Passive diffusion Facilitated diffusion Active transport Group translocation carrier proteins Non Yes transport speed Slow Rapid against gradient transport molecules No specificity Specificity metabolic energy No need Need Solutes molecules Not changed Changed

Utilization of energy in biosynthetic processes
Biosynthetic processes in the cell also require energy; energy from ATP is used to convert one chemical substance into another and to synthesize complex substances from simpler ones Synthesis of small molecules: Amino acids Amino acids-building block of protein 20 amino acids The microorganism growing in the medium – have all 20 of the amino acids present in the medium If they are not available freely in the medium, the microorganism may have to liberate amino acids from proteins by the action of intracellular and extracellular proteolytic enzymes

Sometimes only a few amino acids are present in the medium, in which case the microbes has to convert other amino acids from the available ones into those that are missing In another instances, the medium may contains only inorganic sources of nitrogen, such as ammonium salts The microorganism then has to synthesize all the required amino acids from these sources of available nitrogen All these processes, the interconversion and biosynthesis of chemical substances, require the expenditure of energy

Eg.: synthesis of amino acid proline from Glutamic acid by E.coli
(Rxn) Synthesis of macromolecules: Structure and biosynthesis of a cell wall peptidoglycan This particular biosynthesis also serves as an example of how polymers are synthesized outside the membrane Synthesis of cell wall components is of interest because polymerization takes place outside the cell membrane by enzymes located on the membrane’s outer surface

bacterial cell walls contain a large, complex peptidoglycan molecule consisting of long polysaccharide chains made of alternating N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) residues Pentapeptide chains are attached to the NAM groups. The polysaccharide chains are connected through their pentapeptides or by interbridges Peptidoglycan synthesis complex process Two carriers involves : -uridine diphosphate (UDP)derivatives - bactroprenol, a lipid carrier, to transport NAG-NAM-pentapeptide units across the cell membrane cross links are formed by transpeptidation

Peptidoglycan Synthesis

Peptidoglycan synthesis is particularly vulnerable to disruption by antimicrobial agents
Inhibition of any stage of synthesis weakens the cell wall and can lead to osmotic lysis Many antibiotics interfere with peptidoglycan synthesis. Eg.: penicillin inhibits the transpeptidation reaction, and bacitracin blocks the dephosphorylation of bactoprenol pyrophosphate

Synthesis of organic cell material in chemoautotrophic bacteria
chemoautotrophic bacteria-utilize CO2 as the sole source of carbon oxidize inorganic nutrients – hydrogen, ammonia, nitrite and thiosulfate to produce metabolic energy (in the form of ATP) and reducing power (in the form of NADPH2) - to reduce CO2 and convert it to organic cell material Eg. for the CO2 fixation by autotrophic bacteria – Calvin cycle

Cyanobacteria, some nitrifying bacteria, and thiobacilli possess carboxysomes
polyhedral inclusion bodies – contain ribulose-1,5-bisphosphate carboxylase site of CO2 fixation or may store the carboxylase and other proteins Understanding the cycle is easiest if the calvin cycle is divided into three phases: carboxylation, reduction, and regeneration

Carboxylation Phase Reduction Phase Regeneration Phase
CO2 fixation is accomplished by the enzyme ribulose 1,5-bisphosphate carboxylase or ribulose bisphosphate carboxylase/oxygenase (rubisco), which catalyzes the addition of CO2 to ribulose 1,5-bisphosphate (RuBP), forming two molecules of 3-phosphoglycerate (PGA) Reduction Phase After PGA is formed by carboxylation, it is reduced to glyceraldehyde 3-phosphate reduction, carried out by two enzymes (phosphoglycerate kinase, is essentially a reversal of a portion of the glycolytic pathway, although the glyceraldehyde 3-phosphate dehydrogenase differs from the glycolytic enzyme in using NADP rather than NAD Regeneration Phase third phase of the Calvin cycle regenerates RuBP and produces carbohydrates such as glyceraldehyde 3-phosphate, fructose, and glucose

The formation of glucose from CO2 may be summarized by the following equation
6CO2 +18ATP+12NADPH+ 12H+ +12H2O glucose + 18ADP+18Pi + 12NADP+ ATP and NADPH are provided by photosynthetic light reactions or by oxidation of inorganic molecules in chemoautotrophs Sugars formed in the Calvin cycle can then be used to synthesize other essential molecules Not high utilization of reducing power and energy in this cycle

Microbial Metabolism

Metabolism - all of the chemical reactions within a living organism
Microbial Metabolism: • Aerobic respiration – Glycolysis – TCA – Electron Transport Chain (ET) • anaerobic bioenergetics -Fermentation Metabolism - all of the chemical reactions within a living organism 1. Catabolism ( Catabolic ) breakdown of complex organic molecules into simpler compounds releases ENERGY 2. Anabolism ( Anabolic ) the building of complex organic molecules from simpler ones requires ENERGY

Basic Concepts Reduction and Oxidation
An atom becomes more reduced when it undergoes a chemical reaction in which it Gains electrons By bonding to a less electronegative atom And often this occurs when the atom becomes bonded to a hydrogen An atom becomes more oxidized when it undergoes a chemical reaction in which it Loses electrons By bonding to a more electronegative atom And often this occurs when the atom becomes bonded to an oxygen

In metabolic pathways, we are often concerned with the oxidation or reduction of carbon Reduced forms of carbon (e.g. hydrocarbons, methane, fats, carbohydrates, alcohols) carry a great deal of potential chemical energy stored in their bonds. Oxidized forms of carbon (e.g. ketones, aldehydes, carboxylic acids, carbon dioxide) carry very little potential chemical energy in their bonds.

Reduction and oxidation always occur together. In a reduction-oxidation reaction (redox reaction), one substance gets reduced, and another substance gets oxidized. The thing that gets oxidized is called the electron donor, the thing that gets reduced is called the electron acceptor.

Basic Concepts Enzymatic Pathways for Metabolism
Metabolic reactions take place in a step-wise fashion in which the atoms of the raw materials are rearranged, often one at a time, until the formation of the final product takes place. Each step requires its own enzyme. The sequence of enzymatically-catalyzed steps from a starting raw material to final end products is called an enzymatic pathway (or metabolic pathway)

Basic Concepts Cofactors for Redox Reactions
Enzymes that catalyze redox reactions typically require a cofactor to “shuttle” electrons from one part of the metabolic pathway to another part. There are 2 main redox cofactors: NAD and FAD. These are (relatively) small organic molecules in which part of the structure can either be reduced (e.g., accept a pair of electrons) or oxidized (e.g., donate a pair of electrons)

ATP + H2O ® ADP + Phosphate + Energy (7.5 kcal/mol)
Basic Concepts ATP: A “currency of energy” for many cellular reactions ATP stands for adenosine triphosphate. It is a nucleotide with three phosphate groups linked in a small chain. The last phosphate in the chain can be removed by hydrolysis (the ATP becomes ADP, or adenosine diphosphate). This reaction is energetically favorable: it has a DG°' of about – 7.5 kcal/mol ATP + H2O ® ADP + Phosphate + Energy (7.5 kcal/mol)

ADP + Phosphate + Energy ® ATP + H2O`
Basic Concepts ATP ATP hydrolysis is used as an energy source in many biological reactions that require energy – for example, active transport in the sodium-potassium pump During catabolism, energy released from the oxidation of carbon is captured and used to synthesize ATP from ADP and phosphate. C6H12O6 + 6 O2 ® 6 CO2 + 6 H2O + Energy ADP + Phosphate + Energy ® ATP + H2O`

Microorganisms vary not only in their energy sources, but also in the electron acceptors used by chemotrophs This metabolic process is called respiration and may be divided into 2 different types. aerobic respiration: the final electron acceptor is oxygen, anaerobic respiration: different exogenous acceptor. Most often the acceptor in anaerobic respiration is inorganic (e.g., NO3-, SO42-, CO2, Fe3-,SeO42-, and many others), but organic acceptors such as fumarate may be used. Most respiration involves the activity of an electron transport chain.

Aerobic Cellular Respiration
4 subpathways 1. Glycolysis 2. Transition Reaction 3. Kreb’s Cycle 4. Electron Transport System

1. Glycolysis (splitting of sugar)
Oxidation of Glucose into 2 molecules of Pyruvic acid Embden-Meyerhof Pathway End Products of Glycolysis: 2 Pyruvic acid 2 NADH2 2 ATP 2. Transition Reaction Connects Glycolysis to Krebs Cycle End Products: 2 Acetyl CoEnzyme A 2 CO2 2 NADH2

3. Krebs Cycle (Citric Acid Cycle)
Series of chemical reactions that begin and end with citric acid Products: 2 ATP 6 NADH2 2 FADH2 4 CO2 4. Electron Transport System Occurs within the cell membrane of Bacteria Chemiosomotic Model of Mitchell 34 ATP

How 34 ATP from E.T.S. ? 3 ATP for each NADH2 2 ATP for each FADH2
Glycolysis T. R Krebs Cycle Total 10 x 3 = 30 ATP FADH2 Glycolysis T.R Krebs Cycle Total 2 x 2 = 4 ATP

Total ATP production for the complete oxidation of 1 molecule of glucose in Aerobic Respiration
Glycolysis Transition Reaction Krebs Cycle E.T.S Total ATP

In both aerobic and anaerobic respiration, ATP is formed as a result of electron transport chain activity. 3 stages of aerobic catabolism

1st stage : larger nutrient molecules (proteins, polysaccharides, and lipids) are hydrolyzed or otherwise broken down into their constituent parts such as Amino acids, monosaccharides, fatty acids, glycerol, and other products The chemical reactions occurring during this stage do not release much energy 2nd stage: Amino acids, monosaccharides, fatty acids, glycerol, and other products of the first stage are degraded to a few simpler molecules such as acetyl coenzyme A, pyruvate, and tricarboxylic acid cycle intermediates can operate either aerobically or anaerobically produces some ATP as well as NADH and/or FADH2 3rd stage: nutrient carbon is fed into the tricarboxylic acid cycle during the third stage of catabolism, and molecules are oxidized completely to CO2 with the production of ATP, NADH, and FADH2

The cycle operates aerobically and is responsible for the release of much energy.
Much of the ATP derived from the tricarboxylic acid cycle (and stage-two reactions) comes from the oxidation of NADH and FADH2 by the electron transport chain. Oxygen, or sometimes another inorganic molecule, is the final electron acceptor. These metabolic pathways consist of enzyme catalyzed reactions arranged so that the product of one reaction serves as a substrate for the next.

Embden-Meyerhof or Glycolytic Pathway
most common pathway for glucose degradation to pyruvate in stage two of catabolism. It is found in all major groups of microorganisms and functions in the presence or absence of O2 Glycolysis is located in the cytoplasmic matrix of procaryotes and eucaryotes The glycolytic pathway degrades one glucose to two pyruvates by the sequence of reactions in 2 stages. ATP and NADH are also produced. In the six-carbon stage two ATPs are used to form fructose 1,6- bisphosphate For each glyceraldehyde 3-phosphate transformed into pyruvate, one NADH and two ATPs are formed Because two glyceraldehyde 3-phosphates arise from a single glucose

the three-carbon stage generates four ATPs and two NADHs per glucose
Subtraction of the ATP used in the six-carbon stage from that produced in the three-carbon stage gives a net yield of two ATPs per glucose Thus the catabolism of glucose to pyruvate in glycolysis can be represented by the following simple equation Glucose+2ADP+2Pi+2NAD pyruvate+2ATP+2NADH+2H+

Embden-Meyerhof or Glycolytic Pathway

Tricarboxylic Acid Cycle, or citric acid cycle, or Krebs cycle

Although some energy is obtained from the breakdown of glucose to pyruvate by the glycolytic pathway, much more is released when pyruvate is degraded aerobically to CO2 in stage three of catabolism The multienzyme system called the pyruvate dehydrogenase complex first oxidizes pyruvate to form CO2 and acetyl coenzyme A (acetyl-CoA), an energy-rich molecule composed of coenzyme A and acetic acid joined by a high energy thiol ester bond Acetyl-CoA further degraded in the tricarboxylic acid cycle The substrate for the tricarboxylic acid (TCA) cycle, citric acid cycle, or Krebs cycle is acetyl-CoA

Electron Transport Chain:
The mitochondrial electron transport chain is composed of a series of electron carriers that operate together to transfer electrons from donors, like NADH and FADH2, to acceptors, such as O2

Anaerobic Respiration
Electrons released by oxidation are passed down an E.T.S., but oxygen is not the final electron acceptor Nitrate (NO3-) > Nitrite (NO2-) Sulfate (SO24-) > Hydrogen Sulfide (H2S) Carbonate (CO24-) > Methane (CH4)

Fermentation Anaerobic process that does not use the E.T.S.
Usually involves the incomplete oxidation of a carbohydrate which then becomes the final electron acceptor. Glycolysis - plus an additional step

Fermentation Features of fermentation pathways
Pyruvic acid is reduced to form reduced organic acids or alcohols. The final electron acceptor is a reduced derivative of pyruvic acid NADH is oxidized to form NAD: Essential for continued operation of the glycolytic pathways. O2 is not required. No additional ATP are made. Gasses (CO2 and/or H2) may be released

1. Lactic Acid Fermenation
Only 2 ATP End Product - Lactic Acid Food Spoilage Food Production Yogurt Milk Pickles Cucumbers Sauerkraut - Cabbage 2 Genera: Streptococcus Lactobacillus

2. Alcohol Fermentation Only 2 ATP End products: Alcoholic Beverages
Bread dough to rise Saccharomyces cerevisiae (Yeast)

3. Mixed - Acid Fermentation
Only 2 ATP Escherichia coli and other enterics

4. Propionic Acid Fermentation
Only 2 ATP End Products: Propionic acid CO2 Propionibacterium sp.

Fermentation End Products

Lipid Catabolism

Protein Catabolism

MICROBIAL NUTRITION, GROWTH AND METABOLISM Unit II

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MICROBIAL NUTRITION, GROWTH AND METABOLISM Unit II

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