Microbial Growth PDF

Bacterial Cell Cycle Cell cycle is a sequence of events from formation of new cell through the next cell division...

Bacterial Cell Cycle Cell cycle is a sequence of events from formation of new cell through the next cell division – most bacteria divide by binary fission Microbial Growth: Two pathways function during cycle – DNA replication and partition - Types; Physical factors – cytokinesis - Kinetics; Systems 1 3 Microbial Reproductive Strategies The reproductive strategies of eukaryotic microbes – asexual and sexual, haploid or diploid Bacteria and Archaea – Asexual - binary fission, budding – All must replicate and segregate the genome prior to division 2 4  The Influence of Environmental Extremely Adapted Microbes Factors on Growth Halophiles – grow optimally in the presence of NaCl or Most organisms grow in fairly moderate other salts at a concentration above about environmental conditions 0.2M Extremophiles Extreme halophiles – grow under harsh conditions that would kill – require salt concentrations of 2M and 6.2M most other organisms – extremely high concentrations of potassium – cell wall, proteins, and plasma membrane require high salt to maintain stability and activity 5 7 Microbial response to environmental Effects of NaCl on Microbial conditions Growth Halophiles – grow optimally at >0.2 M Extreme halophiles – require >2 M 6 8  Hydrogen ion concentration (pH) Temperature Microbes cannot regulate their internal temperature The enzymes, electron transport systems and nutrient uptake found in the cell membrane are sensitive to the Enzymes have optimal temperature at which they concentration of hydrogen ions. function optimally Most organisms grow best when H+ and OH- ions are High temperatures may inhibit enzyme functioning present in approximately equal concentrations (pH 7). and be lethal Many bacteria prefer a slightly alkaline medium, while Organisms exhibit distinct cardinal growth many fungi prefer slightly acidic medium. temperatures – minimal The maintenance of a constant pH during microbial – maximal growth is especially important for organisms that – optimal produce acid to avoid self-poisoining 11 pH Temperature The range of temperature for microbial growth can be expressed as three cardinal temperatures: - The minimum temperature is the lowest temperature that permits a microbe’s growth and metabolism, below this temperature its activities are inhibited. - The maximum temperature is the highest temperature at which growth and metabolism can proceed. If the temperature rises slightly above maximum, growth will stop, but if it continues to rise beyond that point, the Acidophiles enzymes and nucleic acids become permanently inactivated and the cell will die. – growth optimum between pH 0 and pH 5.5 - The optimum temperature covers a small range, intermediate between Neutrophiles the minimum and maximum. It promotes the fastest of growth and metabolism during a short period. – growth optimum between pH 5.5 and pH 7 Alkaliphiles (alkalophiles) Depending on their natural habitats, some microbes have a narrow 10 cardinal range, others have a broad one. – growth optimum between pH 8.5 and pH 11.5  Temperature Effect of high temperatures Killing effect of high temperatures (lethal temperature) is Bacteria which can survive exposure to temperature the lowest temperature, at which under certain time are all above the maximum temperature of growth are termed thermoduric bacteria, they are mostly spore microorganisms killed (70 °C/10 min) denaturation of formers. proteins, enzyme inactivation, DNA and cytoplasmatic membrane disruption is dependent on: - species of microorganisms -physiologic status Temperature Ranges for Thermoresistance Microbial Growth psychrophiles – 0o C to 20o C- optimum Degree of microorganisms resistance depends on: 15 or below psychrotrophs – 0o C to 35o C physiologic status of bacteria their genetic properties mesophiles – 20o C to 45o C water content in substrate thermophiles – 55o C to 85o C quantity of protective compounds (lipids, proteins, saccharides) hyperthermophiles – 85o C to 113o C 14  Adaptations of Thermophiles Protein structure stabilized by a variety of means – e.g., more H bonds – e.g., more proline – e.g., chaperones Histone-like proteins stabilize DNA Membrane stabilized by variety of means – e.g., more saturated, more branched and higher molecular weight lipids – e.g., ether linkages (archaeal membranes) 17 19 Basis of Different Oxygen Oxygen Concentration Sensitivities Oxygen easily reduced to toxic reactive oxygen species (ROS) growth in oxygen correlates with microbes energy conserving metabolic processes and – superoxide radical the electron transport chain (ETC) and nature – hydrogen peroxide of terminal electron acceptor – hydroxyl radical Aerobes produce protective enzymes – superoxide dismutase (SOD) – catalase – peroxidase 18 28  Strict Anaerobic Microbes Pressure All strict anaerobic microorganisms lack or have very low quantities of – superoxide dismutase Microbes that live on land and water surface – catalase live at 1 atmosphere (atm) These microbes cannot tolerate O2 Some Bacteria and Archaea live in deep sea Anaerobes must be grown without O2 with very high hydrostatic pressures – work station with incubator – gaspak anaerobic system 21 23 Pressure Barotolerant – adversely affected by increased pressure, but not as severely as nontolerant organisms Barophilic (peizophilic) organisms – require or grow more rapidly in the presence of increased pressure – change membrane fatty acids to adapt to high pressures 24  Radiation Damage Microbial Growth in Natural Environments Ionizing radiation – x-rays and gamma rays Microbial environments are complex, constantly – mutations → death (sterilization) changing, often contain low nutrient – disrupts chemical structure of many concentrations (oligotrophic environment). molecules, including DNA Microbial growth is an autocatalytic process: damage may be repaired by DNA repair mechanisms if small dose no growth will occur without the presence of at least – Deinococcus radiodurans extremely resistant to DNA damage one viable cell and the rate of growth will increase with (prevents oxidative damage, and has the amount of viable biomass present. very potent mechanisms of DNA repair) 25 27 Radiation Damage How do bacteria divide Ultraviolet (UV) radiation – mutations → death – causes formation of thymine dimers in DNA – requires direct exposure on microbial surface – DNA damage can be repaired by several repair mechanisms Growth refers to population growth rather than growth of individual cells 26  Scheme of division 29 Study of 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 Microbiologists usually study population growth rather than growth of individual cells.  Lag phase Stationary growth phase The stationary phase is often due to a growth- limiting Short period immediatelly after inoculation. factor such as the depletion of an essential nutrient, Organisms are synthesising the enzymes and/or the formation of an inhibitory product such as an needed to exploit the new medium. organic acid. Stationary phase results from a situation in which Cell may grow in size but not usually in growth rate and death rate are equal. number. The number of new cells created is limited by the growth If organisms have been transferred from factor and as a result the rate of cell growth matches the an identical medium, at the same rate of cell death. temperature, the lag phase may be very Closed system population growth eventually ceases, total number of viable cells remains constant short. – active cells stop reproducing or reproductive rate is balanced by death rate Log growth phase Death phase At death phase (decline phase), bacteria The log phase (the logarithmic phase or the exponential phase) is a period characterized by cell die. This could be caused by lack of doubling. nutrients, environmental temperature The number of new bacteria appearing per unit time is proportional to the present population. above or below the tolerance band for the If growth is not limited, doubling will continue at a species, or other injurious conditions. constant rate so both the number of cells and the rate of population increase doubles with each consecutive time period. Rate of growth and division is constant and maximal. Population is mostly uniform in terms of chemical and physical properties during this phase.  Balanced Growth During log phase, cells exhibit balanced growth – cellular constituents manufactured at constant rates relative to each other. 39 The Growth Curve Possible Reasons for Stationary Observed when microorganisms are cultivated in Phase batch culture Usually plotted as logarithm of cell number versus Nutrient limitation time Limited oxygen availability Has four distinct phases Toxic waste accumulation 38 40  Stationary Phase and Starvation The Mathematics of Growth Response Generation (doubling) time Entry into stationary phase due to starvation – time required for the population to double in and other stressful conditions activates size survival strategy – varies depending on species of – morphological changes microorganism and environmental conditions e.g., endospore formation – range is from 10 minutes for some bacteria to – decrease in size, protoplast shrinkage, and several days for some eukaryotic nucleoid condensation microorganisms. – Population is doubling every generation 41 43 Senescence and Death Phase The Batch Reactor Two alternative hypotheses Many biochemical processes involve batch growth of cell – cells are Viable But Not Culturable (VBNC) populations. A limited supply of nutrients for growth is provided; when these are used up, or another factor cells alive, but dormant, capable of new growth when becomes limiting, the culture declines. Cells, or products conditions are right. that the organisms have made, can then be harvested from Programmed cell death the culture. After seeding a liquid medium with an inoculums of – fraction of the population genetically programmed to living cell, nothing is added to the culture or die (commit suicide). removed from it as growth proceeds. – Viable but non culturable (VBNC) bacteria refers as to In such a reactor, concentrations of the nutrients, cells bacteria that are in a state of very low metabolic activity and and products vary with time as the growth proceeds. do not divide, but are alive and have the ability to become culturable once resuscitated. 42  Cultivation vessels The Continuous Culture of Microorganisms 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 Scale-up 47 Fed-batch culture (Semi-batch) Importance of Continuous Fed-batch culture is, in the broadest sense, defined as an Culture Methods operational technique in biotechnological processes where one or more nutrients (substrates) are fed (supplied) to the bioreactor Constant supply of cells in exponential during cultivation and in which the product(s) remain in the phase growing at a known rate. bioreactor until the end of the run. An alternative description of the method is that of a culture in which Commonly used in Food and industrial "a base medium supports initial cell culture and a feed medium is microbiology added to prevent nutrient depletion. It is also a type of semi-batch culture. In some cases, all the nutrients are fed into the bioreactor. Fed-batch culture is superior to conventional batch culture when controlling concentrations of a nutrient (or nutrients) affect the yield or productivity of the desired metabolite. Note: the volume of culture liquid is increasing. 48  Batch/Fed-Batch and Continuous culture Fermentor: an apparatus that maintains optimal conditions for the growth of microorganisms, used in large-scale fermentation Chemostat – continuous culture Control flow rate and concentration of growth- limiting nutrient of liquid medium entering and exiting a growth chamber (bioreactor). – Control of: pH Temperature Concentration of terminal electron acceptor Concentration of toxic by-products of metabolism A completely mixed continuous stirred-tank reactor for the cultivation of cells are called chemostats.  Primary and secondary metabolites are often used in industrial microbiology for the production of food, amino Primary metabolites acids, and antibiotics Primary metabolites are considered essential Small molecules of living cells. to microorganisms for proper growth. Intermediates or end products of the Secondary metabolites do not play a role in pathway. growth, development, and reproduction, and are formed during the end or near the stationary Related to synthesis of microbial cells in phase of growth. the growth phase. These metabolites can be used in industrial Include alcohols, amino acids, nucleotides, microbiology to obtain amino acids, develop organic acids, vitamins, and enzymes. vaccines and antibiotics, and isolate chemicals necessary for organic synthesis. Secondary metabolites Accumulate following active growth Have no direct relationship to synthesis of cell material and natural growth Include antibiotics and toxins