Source : Prescott et al Microbiology, Microbiology by Pelczar, Brock Et al Microbiology Microbial Nutrition • Purpose

To obtain energy and construct new cellular components

• Nutrient Requirement

The major elements: C, O, H, N, S, P

The minor elements: K, Ca, Mg, Fe

The trace elements: Mn, Zn, Co, Mo, Ni, Cu Factors affecting growth: Nutritional Factors

Nutrients – Chemicals taken in and used by organisms for energy, metabolism and growth

. Water (Hydrogen and Oxygen) . Carbon . Nitrogen . Sulfur . Phosphorus . Trace Elements . Growth Factors Factors affecting growth: Nutritional Factors

Macronutrients - Required in large amounts

. Carbon . Needed for synthesis of cellular material and energy source . Nitrogen . Needed for protein synthesis, nucleic acids, ATP . Sulfur . Needed to synthesize amino acids and vitamins (thiamine, biotin) . Phosphorus . Needed to synthesize nucleic acids, ATP, phospholipids

Factors affecting growth: Nutritional Factors Trace Elements . required in trace amounts . involved in enzyme function and protein structure . Examples: Zn, Cu, Fe . Present in tap water and distilled

Growth factors . Organic compounds that cannot be synthesized by bacteria . Bacteria are “fastidious” (require relatively large amounts of growth factors in the media. Can be used to test samples for presence of growth factors ) . Examples: amino acids, purines, pyrimidines, vitamins Micronutrients Sources of Essential Nutrients

• Carbon – obtain in organic form, or reduce CO2

-- - • Nitrogen – Fix N2 or obtain as NO3 NO2 , or NH3 • Oxygen – Atmospheric or dissolved in water

• Hydrogen – Minerals, water, organic compounds

• Phosphorous – Mineral deposits

• Sulfur – Minerals, H2S

• Metal Ions - Minerals Make it, or eat it?

• Some bacteria are remarkable, being able to make all the organic compounds needed from a single C source like glucose.

• For others: – Vitamins, amino acids, blood, etc. added to a culture medium are called growth factors.

– Bacteria that require a medium with various growth factors or other components and are hard to grow are referred to as fastidious. Nutrient Requirements

• Prototrophs vs. Auxotrophs – Prototroph – A species or genetic strain of microbe capable of growing on a minimal medium consisting a simple carbohydrate or CO2 carbon source, with inorganic sources of all other nutrient requirements

– Auxotroph – A species or genetic strain requiring one or more complex organic nutrients (such as amino acids, nucleotide bases, or enzymatic cofactors) for growth Carbon sources

Organisms are categorized into two groups:

Autotrophs . Those using an inorganic carbon source (carbon dioxide)

Heterotrophs . Those catabolizing organic molecules i.e. reduced, preformed organic molecules (proteins, carbohydrates, amino acids, and fatty acids)  Phytosynthetic bacteria: Autotrophs Few purple sulphur (e.g., Chromatium) bacteria possess pigments, such as, purple pigment, the bacteriopurpurin, and green pigment, the bacterial chloroyhyll etc. Bacterioviridin occurs hi green sulphur bacteria, e.g., Chlorobium. Such bacteria synthesize their carbohydrate food in presence of sunlight by photosynthesis and are known as chlorophyll bacteria.

2H2S + CO2 → (CH2O)2 + 2S + H2O

 Chemosynthetic bacteria: These bacteria get their energy for food synthesis from the oxidation of certain inorganic chemicals. Light energy is not used. The energy obtained from the chemical reactions is exothermic. The Chemosynthetic bacteria are of the following types:

(a) Sulphomonas (Sulphur bacteria): These bacteria get their energy by oxidation of hydrogen sulphide into H2SO4, e.g., Thiobacillus, Beggiatoa.

CO2 + 2H2S → 2S + H2O + CH2O + Energy

3CO2 + 2S + 8H2O → 2 H2S04 + 2(CH0) + 3H2O + Energy (b) Hydromonas (Hydrogen bacteria): These convert hydrogen into water, e.g., Bacillus pantotrophus.

H2 + ½O2 ® → H2O + Energy (c) Ferromonas (Iron bacteria): These bacteria get their energy by oxidation of ferrous compounds into ferric forms,. e.g., Leptothrix.

2Fe(HCO3)2 + H2O + O → 2Fe (OH)3 + 4CO2 + Energy

4FeCO3 + O2 + 6 H2O → 4Fe(OH)3 + 4CO2 + Energy (d) Methanomonas (Methane bacteria): These bacteria get their energy by oxidation of methane into water and carbon dioxide. (e) Nitrosomonas (Nitrifying bacteria): These bacteria get their energy by oxidation of ammonia and nitrogen compounds into

nitrates. Nitrosomonas oxidises NH3 to nitrites.

NH3 + ½O2 ® → H2O + HNO2 + Energy Nitrobacter converts nitrites to nitrates.

NO2 + ½O2 → NO2 + Energy Heterotrophs Saprophytic bacteria: These bacteria obtain their food from the dead organic decaying substances such as leaves, fruits, vegetables, meat, animal faeces, leather, humus etc. They secrete enzymes to digest the food and absorb it. The breakdown of carbohydrates is fermentation and of proteins the putrefaction. The former produces alcohols, acetic and other organic acids by fermentation of carbohydrates.

Putrefaction decomposes proteins into ammonia, methane, H2S, carbonic acids. The enzymes secreted break down the complex compounds into simpler soluble compounds, which are easily absorbed. Examples are Bacillus acidi lacti, Acetobacter etc. Parasitic bacteria: These bacteria obtain their food from the tissues of living organisms, the hosts. They may be harmless or may cause serious diseases. The disease-producing bacteria are pathogenic which cause various diseases in plants and animals. Examples are Bacillus typhosus, B. anthracis, B. tetani. B. diplheriae, B. tuberculosis, B. pneumoniae, Vibrio cholerae, Pseudomonas citri etc. Symbiotic bacteria: These bacteria live in close association with other organisms as symbionts. They are beneficial to the organisms. The common examples are the nitrogen-fixing bacteria, e.g., Bacillus radicicola, B. azotobacter, Rhizobium, Ctostridium etc. Rhizobium spp.,B. radicicola and B. azotobacter live inside the roots of leguminous plants and form bacteria nodules for fixation of nitrogen from the air. Energy sources

Organisms are categorized into two groups:

Chemotrophs . Acquire energy from redox reactions (oxidation of chemical compounds) involving inorganic and organic chemicals

Phototrophs . use light as their energy source Groups of organisms based on carbon and energy source

Figure 6.1 Electron sources Lithotrophs

. use reduced inorganic substances as their electron source.

Organotrophs

. extract electrons from organic compounds. Nutritional classes based on primary sources of carbon, energy and electrons:

• Phtotolithotrophic autotrophs or photoautotrophs or photolithoautotrophs Source of energy – light energy Source of electrons – Inorganic hydrogen/ electron

Carbon source - CO2 Example: Algae, purple and green sulfur bacteria and cyanobacteria.

• Photoorganotrophic heterotrophy or photoorganoheterotrophy Source of energy – light energy Source of electrons – organic hydrogen/ electron

Carbon source –organic carbon sources (CO2 may also be used) Example: Purple and green nonsulfur bacteria (common inhabitants of lakes and streams) • Chemolithotrophic autotrophs or chemolithoautotrophy Source of energy – Chemical energy source (inorganic) Source of electrons – Inorganic hydrogen/ electron donor

Carbon source - CO2 Example: Sulfur-oxidizing bacteria, hydrogen bacteria, nitrifying bacteria, iron-oxidizing bacteria.

• Chemoorganotrophic heterotrophs or chemoorganoheterotrophy Source of energy – Chemical energy source (organic) Source of electrons – Inorganic hydrogen/ electron donor Carbon source – organic carbon source Example: Protozoan, fungi, most non-photosynthetic bacteria (including most pathogens) Microbial Growth

 Metabolism Results in Reproduction  Reproduction results in Growth

• What is microbial growth? – an increase in a population of microbes (rather than an increase in size of an individual) • Result of microbial growth? – a discrete colony – an aggregation of cells arising from single parent cell Mathematics of Population Growth Mathematics of Population Growth n Nt = No + 2

Number of generations (n) = (log Nt – log No) / log 2 Growth Rate Constant (k) = n/t It is expressed in units of generations per hours (h-1) Generation time (g) = 1/k; it is expressed in units of hours (h). Exponential Growth by Binary Fission

1. DNA replication 2. Cell elongation 3. Septum formation 4. Septum completion leads to separation or further division 5. Process repeats

.Generation time (g= t/n)

.Duration of each division .Determined by type of bacteria .Example: E. coli (20 min) Bacterial Growth Curve The Population Growth Curve

In laboratory studies, populations typically display a predictable pattern over time – growth curve.

Stages in the normal growth curve: 1.Lag phase – “flat” period of adjustment, enlargement; little growth

2.Exponential growth phase – a period of maximum growth will continue as long as cells have adequate nutrients and a favorable environment

3.Stationary phase – rate of cell growth equals rate of cell death caused by depleted nutrients and O2, excretion of organic acids and pollutants

4.Death phase – as limiting factors intensify, cells die exponentially in their own wastes Stationary Phase What – metabolically active cells stop reproducing – reproductive rate is balanced by death rate • Why – nutrient limitation – limited oxygen availability – toxic waste accumulation – critical population density reached • Starvation Response – Morphological change – Decrease in cell size – Production of starvation proteins Diauxic growth

• Growth in two phases • Utilize one carbon source first • Utilize the second one until the first one depleted • Resulted from inducible enzyme synthesis Environmental Effects on Bacterial Growth

• Temperature

• Oxygen

• pH

• Osmotic pressure Temperature Cardinal temperatures • Minimum Temperature: Temperature below which growth ceases, or lowest temperature at which microbes will grow.

• Optimum Temperature: Temperature at which growth rate is the fastest.

• Maximum Temperature: Temperature above which growth ceases, or highest temperature at which microbes will grow. Classification of Microorganisms by Temperature Requirements Temperature Classes of Organisms • Psychrophiles ( 00C-200C) – Cold temperature optima – Most extreme representatives inhabit permanently cold environments

• Mesophiles ( 200C – 450C) – Midrange temperature optima – Found in warm-blooded animals and in terrestrial and aquatic environments in temperate and tropical latitudes

• Thermophiles ( 500C- 800C) – Growth temperature optima between 45ºC and 80ºC

• Hyperthermophiles – Optima greater than 800C – These organisms inhabit hot environments including boiling hot springs, as well as undersea hydrothermal vents that can have temperatures in excess of 100ºC Temperature Psychrotrophs and Mesophiles Growth vs. Tolerance – “Growth” is generally used to refer to the acquisition of biomass leading to cell division, or reproduction – Many microbes can survive under conditions in which they cannot grow – The suffix “-phile” is often used to describe conditions permitting growth, whereas the term “tolerant” describes conditions in which the organisms survive, but don’t necessarily grow – For example, a “thermophilic bacterium” grows under conditions of elevated temperature, while a “thermotolerant bacterium” survives elevated temperature, but grows at a lower temperature 34 Use of Temperature to Preserve Microbes Preserving Bacteria Cultures: • Refrigeration: – Storage for short periods of time

• Deep-freezing: – -50° to -95°C – Preserves cultures for years

• Lyophilization (freeze-drying): – Frozen (-54° to -72°C) and dehydrated in a vacuum – Can last decades Oxygen Requirements Oxygen sources

. Found as gaseous O2 or covalently bound in compounds . Essential for aerobic respiration . Oxygen is the final electron acceptor • Deadly for some types of bacteria (anaerobes)

. Toxic forms of oxygen are highly reactive . are excellent oxidizing agents . results in irreparable damage to cells by oxidizing compounds such as proteins and lipids Classification of organisms based on O2 utilization

• Obligate (strict) aerobes require O2 in order to grow, Ex. Bacillus, Pseudomonas

• Obligate (strict) anaerobes cannot survive in O2 , Ex. Clostridium sp. Facultative anaerobes grow better in O2,Ex. E. coli, Staphylococcus

• Aerotolerant organisms don’t care about O2 ,Ex. Lactobacillus sp.

• Microaerophiles require low levels of O2

 Capnophile – requires higher CO2 tension (3-10%) than normally found in the atmosphere, Ex. Neisseria, Brucella, S. pneumoniae 38 Obligate (strict) vs. facultative – “Obligate” (or “strict”) means that a given condition is required for growth

– “Facultative” means that the organism can grow under the condition, but doesn’t require it • The term “facultative” is often applied to sub-optimal condition

– For example, an obligate thermophile requires elevated temperatures for growth, while a facultative thermophile may grow in either elevated temperatures or lower temperatures OxygenToxicity

Hydrogen peroxide

Superoxide

Hydroxyl radical (OH) •Result of ionizing radiation & incomplete reduction of hydrogen peroxide; extremely reactive but danger averted in aerobes because of catalase & peroxidase Oxygen Toxicity

41 Effects of pH

• Classification of Microbes based on pH – Organisms sensitive to changes in acidity – H+ and OH– interfere with H bonding

– Acidophiles – prefer below 7 – Neutrophiles – prefer 7 – Alkalinophiles – prefer above 7

– Most bacteria grow between pH 6.5 and 7.5 – Molds and yeasts grow between pH 5 and 6 Physical Effects of Water .Microbes require water . to dissolve enzymes and nutrients required in metabolism; to react in many metabolic reactions . Some microbes have cell walls that retain water . Endospores and cysts stop most metabolic activity to survive in a dry environment for years

. Two physical effects of water . Osmotic pressure . Hydrostatic pressure Osmotic Pressure Osmotic pressure The pressure exerted on the semipermeable membrane by a solution containing solutes, which cannot move across the membrane.

Osmosis Diffusion of water across a semipermeable membrane driven by unequal concentration of solutes across the membrane. Osmotic Pressure

ISOTONIC HYPERTONIC Physiologic Saline Osmotic Variations in the Environment – Isotonic – External concentration of solutes is equal to cell’s internal environment – Diffusion of water equal in both directions – No net change in cell volume – Hypotonic – External concentration of solutes is lower than cell’s internal environment – Cells swell and burst – Hypertonic – Environment has higher solute concentration than cell’s internal environment – Cells shrivel (crenate) – Halophiles tolerate higher salt concentrations Hydrostatic Pressure .Water exerts pressure in proportion to its depth . For every addition of depth, water pressure increases 1 atm .Organisms that live under extreme pressure are barophiles . Their membranes and enzymes depend on this pressure to maintain their three-dimensional, functional shape Culture Media MEDIA • Nutrient preparation for microbial growth • Must provide all chemical requirements • Physical state (Broth-liquid, Agar-Solid) AGAR – Used as solidifying agent for culture media (Typically 1.5-2.0%) – Composed of complex polysaccharides – Advantages of agar vs gelatin: – Generally not metabolized by microbes – Liquefies at 100°C – Solidifies ~40°C . Fanny Hesse used agar from seaweed(Red Algae) in her jams and jellies, which she learned from a neighbor who had lived in Java (Indonesia). Types of Media Used  Defined medium : precise amounts of highly purified chemicals  Complex medium (or undefined) : highly nutritious substances. . Basic Nutrient . Designed to grow broad-spectrum microbes . Enriched . Add enrichment to encourage growth of microbes . Blood, growth factors, serum . Selective . Suppress unwanted microbes and encourage desired microbes to grow . Salt, dyes, alcohol . Differential . To distinguish colonies of different microbes from one another . Dyes, pH indicators . Reduced (anaerobic) media

. Contain chemicals (thioglycollate) that combine O2, Used for anaerobic cultures Chemically Defined vs Complex Media Selective medium MacConkey agar as a selective and differential medium Anaerobic Culture Methods

Gas Pak Jar Glove Box Capnophiles require high CO2

• Candle jar

(3-10% CO2)

• CO2-packet Planktonic vs Sessile Bacteria

•All lab tests use “pure cultures” of suspended cells called planktonic bacteria since they float around in liquid.

•In fact, pure cultures are virtually absent in nature.

•Most microbes exist as sessile

Robert Koch bacteria– attached to a surface – and they live in communities called biofilms. Biofilms . An organized, layered system of microbes attached to a surface . Biofilms form when microbes adhere to a surface that is moist and contains organic matter – Complex relationships among numerous microorganisms

– Develop an extracellular matrix – Adheres cells to one another – Allows attachment to a substrate – Sequesters nutrients – May protect individuals in the biofilm

– Form on surfaces often as a result of quorum sensing

– Many microorganisms more harmful as part of a biofilm How does a biofilm develop?

1. Planktonic cells attach to surface 2. Cells multiply ;Produce glycocalyx 3. Slime layer entraps nutrients, cells, microbes 4. Dynamic pillar-like layers form How do biofilms communicate?

•Cell to cell communication - send and receive chemical signaling molecules

•Quorum sensing - accumulation of signaling molecules

- enables a cell to sense the cell density Where are Biofilms Found? Biofilms Found in Health Care

. Dental caries

. Contact lenses

. Lungs of Cystic Fibrosis patients Biofilm on a contact lens

. Indwelling medical devices . Endotracheal tube . Mechanical heart valves . Pacemakers . Urinary catheters . IV connectors Staphylococcus biofilm . Prosthetic joints on inner surface of IV connector Medical Importance of Biofilms

.Are 1000X more resistant to antimicrobial agents than planktonic cells .Easily transfer genes to express new and sometimes more virulent phenotypes .Are more resistant to host defense mechanisms

.80% of nosocomial infections are biofilm associated (NIH) .20% of patients with biofilm-related septicemia die Quorum Sensing • A mechanism by which members of a bacterial population can behave cooperatively, altering their patterns of gene expression (transcription) in response to the density of the population

• In this way, the entire population can respond in a manner most strategically practical depending on how sparse or dense the population is. Mechanism: • As the bacteria in the population grow, they secrete a quorum signaling molecule into the environment (for example, in many gram-negative bacteria the signal is an acyl homoserine lactone, HSL) • When the quorum signal reaches a high enough concentration, it triggers specific receptor proteins that usually act as transcriptional inducers, turning on quorum-sensitive genes