Chapter 6 Microbial Nutrition and Growth
Growth Requirements Microbial growth Increase in a population of microbes Due to reproduction of individual microbes Result of microbial growth is discrete colony An aggregation of cells arising from single parent cell
Growth Requirements Organisms use a variety of nutrients for their energy needs and to build organic molecules and cellular structures Most common nutrients contain necessary elements such as carbon, oxygen, nitrogen, and hydrogen Microbes obtain nutrients from variety of sources
Microbial nutrition Nutrition process by which chemical substances (nutrients) are acquired from the environment and used in cellular activities Essential nutrients must be provided to an organism Essential nutrients Macronutrients required in large quantities play principal roles in cell structure and metabolism Proteins, carbohydrates Micronutrients or trace elements required in small amounts involved in enzyme function and maintenance of protein structure Manganese, Nickel,Magnesium, Zinc
Nutrients Organic nutrients contain carbon and hydrogen atoms usually the products of living things Methane (CH 4 ), carbohydrates, lipids, proteins, nucleic acids Inorganic nutrients atom or molecule that contains a combination of atoms other than carbon and hydrogen Metals and their salts (magnesium sulfate, ferric nitrate, sodium phosphate), gases (oxygen, carbon dioxide), water
Chemical analysis of cell contents 70% water Proteins- most prevalent organic compounds 96% of cell is composed of 6 elements: CHONPS Carbon Hydrogen Oxygen Nitrogen Phosphorous Sulfur
Growth factors: Essential organic nutrients Organic compounds that cannot be synthesized by an organism because they lack the genetic and metabolic mechanisms to synthesize them Growth factors must be provided as a nutrient Essential amino acids, vitamins Also look at Table 6.1
Growth factors: Essential organic nutrients Haemophilus influenzae is a fastidious organism Grows best at 35-37 C with ~5% CO 2 (or in a candle-jar) Requires Hemin (X factor) Nicotinamide-adenine-dinucleotide (NAD) also known as V factor Chocolate agar plate (CAP) Prepared with heat-lysed horse/sheep blood: good source of both hemin and NAD NAD is released from the blood during the heating process Hemin is available from non-hemolyzed as well as hemolyzed blood cells. www.cdc.gov
Nutrients: Chemical and Energy Requirements Two groups of organisms based on source of carbon Autotrophs Heterotrophs Two groups of organisms based on source of energy Chemotrophs Phototrophs Two groups of organisms based on source of electrons Organotrophs Lithotrophs
Four basic groups of organisms based on their carbon and energy sources Figure 6.1
Heterotrophs and their energy sources Majority are chemoheterotrophs Aerobic respiration Two categories Saprobes: free-living microorganisms that feed on organic detritus from dead organisms Opportunistic pathogen Facultative parasite Parasites: derive nutrients from host Pathogens Some are obligate parasites
Parasites Facultative parasites Saprobes infecting a host Occurs when host is compromised (chemotherapy, AIDS) Opportunistic pathogen Pseudomonas aeruginosa problem in hospitals Obligate parasites Pathogens- can cause disease or even death Ectoparasites: live on the body Endoparasites: live in organs & tissues Intracellular parasites: live in the cells
Oxygen Requirements Oxygen is essential for obligate aerobes Oxygen is deadly for obligate anaerobes How can this be true? Toxic forms of oxygen are highly reactive and excellent oxidizing agents Resulting oxidation causes irreparable damage to cells
Four Toxic Forms of Oxygen Singlet oxygen ( 1 O 2 ) Superoxide radicals (O - 2 ) Hydrogen Peroxide (H 2 O 2 ) Hydroxyl radical (OH - )
Neutralizing toxic forms of Oxygen Most cells have developed enzymes that neutralize these chemicals: Superoxide dismutase Catalase Peroxidase Catalase converts H 2 O 2 to water and oxygen Catalase test useful to distinguish staphylococci from streptococci Figure 6.2
Aerobes Anaerobes Facultative anaerobes Aerotolerant anaerobes Microaerophiles Oxygen requirements Oxygen concentration High Loosefitting cap Figure 6.3 Low Obligate aerobes Obligate anaerobes Facultative anaerobes Aerotolerant anaerobes Using a liquid thioglycollate growth medium to identify the oxygen requirements of organisms.
Categories of oxygen requirement Aerobe utilizes oxygen and can detoxify it Anaerobe does not utilize oxygen Obligate aerobe cannot grow without oxygen Facultative anaerobe utilizes oxygen but can also grow in its absence Obligate anaerobe lacks the enzymes to detoxify oxygen so cannot survive in an oxygen environment Aerotolerant anaerobes do not utilize oxygen but can survive and grow in its presence Microaerophilic requires only a small amount of oxygen
Physical Requirements: Temperature Temperature affects threedimensional structure of proteins Lipid-containing membranes of cells and organelles are temperature sensitive If too low, membranes become rigid and fragile Minimum Optimum Maximum If too high, membranes become too fluid Figure 6.4 22ºC 30ºC 37ºC
Three temperature adaptation groups Psychrophiles optimum temperature below 15 o C; capable of growth at 0 o C Mesophiles optimum temperature 20 o -40 o C; most human pathogens Thermophiles optimum temperature greater than 45 o C Figure 6.5
gure 6.6 An example of a psychrophile in Antartica.
Physical Requirements: ph Organisms sensitive to changes in acidity Neutrophiles grow best in a narrow range around neutral ph Acidophiles grow best in acidic habitats Alkalinophiles live in alkaline soils and water
Physical effects of water Microbes require water to dissolve enzymes and nutrients Water is important reactant in many metabolic reactions Most cells die in absence of water Two physical effects of water Osmotic pressure Hydrostatic pressure
Osmotic pressure Pressure exerted on a semipermeable membrane by a solution containing solutes that cannot freely cross membrane Hypotonic solutions have lower solute concentrations Hypertonic solutions have greater solute concentrations Restricts organisms to certain environments: Obligate and facultative halophiles
Osmotic pressure Most microbes exist under hypotonic or isotonic conditions Halophiles require a high concentration of salt Halobacterium grows in 9-25% NaCl solutions Osmotolerant do not require high concentration of solute but can tolerate it when it occurs Staphylococcus aureus can grow in 0.1-20% NaCl media High salt & sugar concentrations used to preserve food like jellies & brine could support microbial growth
Hydrostatic pressure Water exerts pressure in proportion to its depth Barophiles live under extreme pressure Their membranes and enzymes depend on pressure to maintain their three-dimensional, functional shape
Carbon dioxide requirement All microbes require some carbon dioxide in their metabolism Capnophile grows best at higher CO 2 tensions than normally present in the atmosphere Neiserria sp (gonorrhea, meningitis) Brucella abortus (undulant fever)
Microbial classification by their environmental niche
Ecological associations among microorganisms Microbial Associations Symbiotic Organisms live in close nutritional relationships; required by one or both members. Nonsymbiotic Organisms are free-living; relationships not required for survival Mutualism Obligatory, dependent; both members benefit. Commensalism The commensal benefits; other member not harmed. Parasitism Parasite is dependent and benefits; host harmed. Synergism Members cooperate and share nutrients. Antagonism Some members are inhibited or destroyed by others.
Ecological associations (Symbiotic) Mutualism obligatory, dependent; both members benefit CAP BAP Commensalism commensal member benefits, other member neither harmed nor benefited Parasitism parasite is dependent and benefits; host is harmed Staphylococcus aureus growth Haemophilus satellite colonies
Ecological associations (Non symbiotic) Synergism members cooperate to produce a result that none of them could do alone Antagonism actions of one organism affect the success or survival of others in the same community (competition) Antibiosis- example of antagonism: antibiotics Masahiro Tagawa, et.al. FEMS Microbiology Letters Apr 2010, 305 (2) 136-142
Interrelationships between microbes and humans Human body is a rich habitat for symbiotic bacteria, fungi- normal microbial flora Commensal, parasitic, and synergistic relationships HUMAN MICROBIOME PROJECT (HMP) Researchers in the HMP are sampling and analyzing the genome of microbes from five sites on the human body. http://commonfund.nih.gov/hmp/index https://www.bcm.edu/departments/molecular-virologyand-microbiology/microbiome
Associations and Biofilms Complex relationships among numerous microorganisms Form on surfaces, medical devices, mucous membranes of digestive system Form as a result of quorum sensing Many microorganisms more harmful as part of a biofilm Scientists seeking ways to prevent biofilm formation
Biofilm development 1 Free-swimming microbes are vulnerable to environmental stresses. Bacteria Chemical structure of one type of quorum- sensing molecule Water flow Water channel Escaping microbes Matrix Some microbes 2 land on a surface, 3 such as a tooth, and attach. The cells begin producing an intracellular matrix and secrete quorumsensing molecules. Quorum sensing New cells arrive, triggers cells to possibly including 4 change their 5 6 new species, and biochemistry and water channels form shape. in the biofilm. Some microbes escape from the biofilm to resume a free-living existence and perhaps, form a new biofilm on another surface. Figure 6.7
Rajesh Ramakrishnan, Masters Thesis
1 2 3 4 Cytoplasmic membrane Chromosome Cell wall Replicated chromosome Septum Completed septum Binary fission 30 minutes Figure 6.17 5 60 minutes Septum 90 minutes 120 minutes Generation Time: Time required for a bacterial cell to grow and divide
Bacteria show logarithmic (exponential) growth Figure 6.18
A typical microbial growth curve In laboratory studies, populations typically display a predictable pattern over time growth curve Figure 6.20
The population growth curve Stages in the normal growth curve: 1) Lag phase flat period of adjustment, enlargement; little growth
Stages in the normal growth curve: 1) Lag phase The population growth curve 2) Exponential growth phase a period of maximum growth will continue as long as cells have adequate nutrients and a favorable environment
The population growth curve Stages in the normal growth curve: 1) Lag phase 2) Exponential growth phase 3) Stationary phase rate of cell growth equals rate of cell death caused by depleted nutrients and O 2, excretion of organic acids and pollutants
The population growth curve Stages in the normal growth curve: 1) Lag phase 2) Exponential growth phase 3) Stationary phase 4) Death phase as limiting factors intensify, cells die exponentially
Direct Measuring Microbial Reproduction Indirect not requiring incubation 1) Microscopic counts 2) Electronic counters requiring incubation 1) Serial dilution 2) Viable plate counts 3) Membrane filtration 4) Most probable number (MPN) 1) Turbidity 2) Dry weight 3) Genetic methods
The use of a cell counter for estimating microbial numbers. Cover slip Pipette Bacterial suspension Location of grid Overflow troughs Place under oil immersion Coulter counters Bacterial suspension Flow cytometry Figure 6.22
A serial dilution and viable plate count for estimating microbial population size 1 ml of original culture 1.0 ml 1.0 ml 1.0 ml 1.0 ml 9 ml of broth + 1 ml of original culture 1:10 dilution (10-1 ) 1:100 dilution (10-2 ) 1:1000 dilution (10-3 ) 1:10,000 dilution (10-4 ) 1:100,000 dilution (10-5 ) 0.1 ml of each transferred to a plate 0.1 ml 0.1 ml 0.1 ml 0.1 ml Incubation period Too numerous to count (TNTC) TNTC 65 colonies 6 colonies 0 colonies Choose a plate that appears to have between 30 and 300 colonies.
Mathematical reasoning for performing the serial dilutions Tube 1 contains 9 ml of sterile media; you will add 1 ml of the undiluted bacterial suspension to yield a total volume of 10 ml. 1 1 1 x 10-1 1:10 dilution 9 ml + 1 ml 10 ml Tube 2 contains 9 ml of sterile media; you will add 1 ml of the 1:10 diluted bacterial suspension to yield a total volume of 10 ml 1 1 x 1 9 ml + 1 ml 10 ml 10 ml 1 x 10-2 1:100 dilution
The use of membrane filtration to estimate microbial population size. Sample to be filtered Membrane transferred to culture medium Membrane filter retains cells To vacuum Colonies Incubation Figure 6.24
The most probable number (MPN) method for estimating microbial numbers. 1.0 ml 1.0 ml Undiluted 1:10 1:100 Inoculate 1.0 ml into each of 5 tubes Phenol red, ph color indicator, added Incubate Results 4 tubes positive 2 tubes positive 1 tube positive
Turbidity and the use of spectrophotometry in indirectly measuring population size. Direct light Light source Uninoculated tube Light-sensitive detector Light source Inoculated Scattered light broth culture that does not reach reflector Figure 6.26
Other Indirect methods of measuring microbial growth Metabolic activity Dry weight Genetic methods Isolate DNA sequences of unculturable prokaryotes