BIOL 3340 Chapter 7

Chapter 7 Microbial Growth

Growth Increase in cellular constituents that may result in: increase in cell number e.g., when microorganisms reproduce by budding or binary fission increase in cell size e.g., coenocytic microorganisms have nuclear divisions that are not accompanied by cell divisions e.g aseptic fungi microbiologists usually study population growth rather than growth of individual cells Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

The Procaryotic Cell Cycle Cell cycle is sequence of events from formation of new cell through the next cell division most bacteria divide by binary fission Two pathways function during cycle DNA replication and partition cytokinesis Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Figure 7.1: Binary Fission Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Chromosome Replication and Partitioning Most procaryotic chromosomes are circular Origin of replication – site at which replication begins Terminus – site at which replication is terminated & is located directly opposite the origin Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

..Growth Replisome: Replisome is a group of proteins needed for DNA synthesis which assembled at the origin. DNA replication then proceeds in both directions. As daughter chromosomes are synthesised, the 2 newly formed origins moves towards opposite ends of the cell Copyright © McGraw-Hill companies, Inc. Permission required for reproduction or display.

Figure 7.2:Cell Cycle of E.coli Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Figure 7.3-Cytoskeletal protein such as Actin homologue MreB , tubulin protein FTsZ & MinCD Min protein. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Cytoskeletal Proteins − Role in Cytokinesis Cytokinesis is process not well understood Protein MreB similar to eucaryotic actin plays a role in determination of cell shape and movement of chromosomes to opposite cell poles Protein FtsZ similar to eucaryotic tubulin plays a role in Z ring formation which is essential for septation MinCD-Min protein oscillates from pole to pole preventing an offcentre Z ring. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

…Growth Cytokinesis is completed in several steps: 1. Selection of site where septum is formed 2. Assembly of the specialized structure, the Z ring which divides the cell into 2 by constriction 3. Linkage of Z ring to plasma membrane and may be other components of cell wall

..Growth 4. Assembly of cell wall synthesizing machinery (e;g of peptidoglycan and other cell wall materials 5. Constriction of the cell wall accompanied by invagination of the cell membrane and septum formation. Assembly of Z ring is crucial for other steps to follow. Copyright © McGraw-Hill companies, Inc. Permission required for reproduction or display.

DNA Replication in Rapidly Growing Cells A cell cycle is completed in 60 minutes e.g is a slow growing E coli. 40 minutes for DNA replication 20 minutes for septum formation and cytokinesis Rapidly growing E. coli, a cell cycle may be completed in 20 mins. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

The Growth Curve A growth curve is observed when microorganisms are cultivated in batch culture A culture incubated in a closed vessel with a single batch of medium usually plotted as logarithm of cell number versus time usually has four distinct phases Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

..Growth Curve Typically, a batch culture passes through four distinct stages: Lag stage Logarithmic (exponential) growth Stationary stage Death stage Copyright © McGraw-Hill companies, Inc. Permission required for reproduction or display.

Figure 7.5: Microbial Growth Curve in a closed system Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Lag Phase Cell synthesizing new components Varies in length e.g., to replenish spent materials e.g., to adapt to new medium or other conditions E.g cells may be old, synthesizes ATP, essential enzymes, ribosomes etc..or injured need time to recover Varies in length in some cases can be very short or even absent Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Exponential Phase Also called log phase Rate of growth is constant and also called as balanced growth Population is most uniform in terms of chemical and physical properties during this phase Exponential growth- all cellular components are synthsized at constant rate Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Unbalanced Growth Rates of synthesis of cell components vary relative to each other occurs under a variety of conditions change in nutrient levels shift-up (poor medium to rich medium) shift-down (rich medium to poor medium) change in environmental conditions Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Balanced Growth During log phase, cells exhibit balanced growth cellular constituents manufactured at constant rates relative to each other Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Effect of Nutrient Concentration on Growth Figure 7.6 a): Nutrient concentration on total yield of microbial cells b) Nutrient on Growth rate Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

….Growth Curve Few similarities to passive diffusion: Movement of substances across a membrane with the assistance of a transport protein movement of molecules is not energy dependent direction of movement is from high concentration to low concentration Copyright © McGraw-Hill companies, Inc. Permission required for reproduction or display.

Stationary Phase Total number of viable cells remains constant during lag phase may occur because metabolically active cells stop reproducing may occur because reproductive rate is balanced by death rate Nutrient depletion e.g oxygen Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Possible Reasons for Entry into Stationary Phase Nutrient limitation Limited oxygen availability Toxic waste accumulation Critical population density reached Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Active Transport Energy-dependent process ( ATP) Movement of substances across a membrane with the assistance of a transport protein (permeases).Active transport pumps are usually carrier proteins. Energy-dependent process ( ATP) Moves molecules against the concentration gradient i.e Movement of solutes from low  high concentration Concentrates molecules inside cell. E.g ATP/ABC Transporters Copyright © McGraw-Hill companies, Inc. Permission required for reproduction or display.

Starvation Responses morphological changes e.g., endospore formation decrease in size, protoplast shrinkage, and nucleoid condensation production of starvation proteins which makes cells much more resistant to damage. E;g increases in peptidodoglycan or DNA binding protein to protect DNA. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

…Starvation Responses Protein Chaperone- to prevent protein denaturation Hence starved cells difficult to kill, long-term survival ( for years) & increased virulence e.g Salmonella enterica VBNC Viable but non-Culturable

Loss of Viability Figure 7.7: Loss of Viability Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

The Mathematics of Growth Generation (doubling) time time required for the population to double in size varies depending on species of microorganism and environmental conditions range is from 10 minutes for some bacteria to several days for some eucaryotic microorganisms Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Figure 7.9: Exponential Microbial growth: Four generations of growth (red) plotted directly & in the logarithmic form (BlUe). Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

The mean generation time (doubling time) is the amount of time required for the concentration of cells to double during the log stage. It is expressed in minutes Growth rate (min-1) = Mean generation time can be determined directly from a semilog plot of bacterial concentration vs time after inoculation

Table 7.1 : 2 cells after 20 mins, 4 cells after 40 mins,, No =initial population number, n is the number of generations & Nt = Incubation time Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Measurement of Microbial Growth can measure changes in number of cells in a population can measure changes in mass of population the most appropriate method depends on the experimental situation Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Measurement of Cell Numbers Direct cell counts: Counting chambers e.g Petroff-Hausser (Fig 7.11) for prokaryotes & Hemocytometers for pro and eucaryotes Electronic counters for larger count such as yeast eg Flow cytometry Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Counting Chambers easy, inexpensive, and quick useful for counting both eucaryotes and procaryotes cannot distinguish living from dead cells Figure 7.11-The Petroff-Hausser Counting Chamber Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Viable cell counts: Plating methods- several plating method can be used to determine the number of viable microbes in a sample e.g spread plate and pour plate. In both methods a diluted sample is dispersed over the media. Membrane filters (fig.7.12)-

Plating Methods… simple and sensitive widely used for viable counts of microorganisms in food, water, and soil inaccurate results obtained if cells clump together Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

..membrane filter In membrane filter method, bacterial cells are trapped from aquatic samples on a membrane filter. Filter is then placed on agar media, incubated until colony is formed Colony count is done

..Viable count Method - Membrane filtration especially useful for analyzing aquatic samples Figure 7.12-The Membrane Filtration procedure Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Figure 7.13-Colonies on membranes Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Measurement of Cell Mass Dry weight time consuming and not very sensitive Quantity of a particular cell constituent e.g., protein, DNA, ATP, or chlorophyll useful if amount of substance in each cell is constant Turbidometric measures (light scattering) quick, easy, and sensitive Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Figure 7.14-Microbial mass Measurement more cells  more light scattered less light detected Figure 7.14-Microbial mass Measurement Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

The Continuous Culture of Microorganisms Batch cultures in a closed system ( so far decribed) are those in which nutrients are replaced nor wastes are removed. In a continous culture, growth in an open system continual provision of nutrients continual removal of wastes maintains cells in log phase at a constant biomass concentration for extended periods achieved using a continuous culture system e.g Chemostat Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

…chemostat A chemostat- the rate at which sterile medium is fed into the culture vessel, is the same as the rate at which media containing microorganisms is removed. Chemostat contains essential nutrient e.g a vitamin in limiting quantities.

The Chemostat rate of incoming medium = rate of removal of medium from vessel an essential nutrient is in limiting quantities Figure 7.15 Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

The Turbidostat regulates the flow rate of media through vessel to maintain a predetermined turbidity or cell density by a photocell. dilution rate varies no limiting nutrient turbidostat operates best at high dilution rates Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

The Influence of Environmental Factors on Growth most organisms grow in fairly moderate environmental conditions Extremophiles grow under harsh conditions that would kill most other organisms Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Solutes and Water Activity water activity (aw): amount of water available to organisms reduced by interaction with solute molecules (osmotic effect) higher [solute]  lower aw reduced by adsorption to surfaces (matric effect) Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

..Water Activity (aw) water activity of a solution is 1/100 the relative humidity of solution also equal to ratio of vapor pressure (Psoln) of the solution to that of pure water (Pwater) Aw = Psoln/ Pwater Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Osmotolerant Organisms grow over wide ranges of water activity many use compatible solutes to increase their internal osmotic concentration compatible solutes are those which compatible with metabolism and growth some have proteins and membranes that require high solute concentrations for stability and activity. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Effects of NaCl on Microbial Growth halophiles grow optimally at >0.2 M, high level of NACL. extreme halophiles require >2 M e.g Halobacterium can grow in Dead Sea salt lake Figure 7.17 Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

pH acidophiles neutrophiles alkalophiles growth optimum between pH 0 and pH 5.5 neutrophiles growth optimum between pH 5.5 and pH 8 alkalophiles growth optimum between pH 8 and pH 11.5 Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

…pH most acidophiles and alkalophiles maintain an internal pH near neutrality some synthesize proteins that provide protection e.g., acid-shock proteins many microorganisms change pH of their habitat by producing acidic or basic waste products most media contain buffers to prevent growth inhibition Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Temperature organisms exhibit distinct cardinal growth temperatures minimal maximal optimal Figure 7.19 Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Figure 7.20 Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Oxygen Concentration Figure 7.21-Oxygen and Bacterial growth:SOD ( Enzyme superoxide dismutase) and catalase. Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Basis of Different Oxygen Sensitivities oxygen easily reduced to toxic products superoxide radical hydrogen peroxide hydroxyl radical aerobes produce protective enzymes superoxide dismutase (SOD) from toxic superoxide radical Catalase-destroy hydrogen peroxide Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Pressure barotolerant barophilic organisms adversely affected by increased pressure, but not as severely as nontolerant organisms barophilic organisms require or grow more rapidly in the presence of increased pressure (400 atm or greater) Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Radiation Damage ionizing radiation x rays and gamma rays mutations  death disrupts chemical structure of many molecules, including DNA damage may be repaired by DNA repair mechanisms Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Radiation Damage… ultraviolet (UV) radiation mutations  death causes formation of thymine dimers in DNA DNA damage can be repaired by several repair mechanisms Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Microbial Growth in Natural Environments microbial environments are complex, constantly changing, often contain low nutrient concentrations (oligotrophic environment) and may expose a microorganism to overlapping gradients of nutrients and environmental factors Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Responses to Low Nutrient Levels (oligotrophic environments) organisms become more competitive in nutrient capture and use of available resources morphological changes to increase surface area and ability to absorb nutrients mechanisms to sequester certain nutrients Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Biofilms ubiquitous in nature complex, slime enclosed colonies attached to surfaces than free floating. when form on medical devices such as implants often lead to illness can be formed on any conditioned surface Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

…Biofilm Microorganisms extracellular matrix and change in attached organisms’ physiology protects them from harmful agents such as UV light and antibiotics sloughing off of organisms can result in contamination of water phase above the biofilm such as in a drinking water system Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Cell to Cell Communication Acylhomoserine lactone (AHL) is an autoinducer molecule produced by many gram-negative organisms e.g marine luminescent Vibrio fisheri which glows. AHL control the ability this bacterium to glow. concentration present allows cells to access population density process is called quorum sensing Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

..Quorum Sensing AHL or other signal molecule diffuses across plasma membrane at high concentrations it enters cell once inside the cell it induces expression of target genes that regulate a variety of functions Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Fig. 7.28-Euprymna scolopes ( squid) buried day time and active at night. Light organ is covered with Vibrio fisheri Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Bibliography Lecture PowerPoints Prescott’s Principles of Microbiology-Mc Graw Hill Co. http://en.wikipedia.org/wiki/Scientific_method https://files.kennesaw.edu/faculty/jhendrix/bio3340/home.html