Industrial Microbiology Bacterial Growth Industrial Microbiology

Bacterial growth Binary fission Generation time Phases of growth 4-2

Binary fission Prokaryote cells grow by increasing in cell number (as opposed to increasing in size). Replication is by binary fission, the splitting of one cell into two Therefore, bacterial populations increase by a factor of two (double) every generation time. Figure 4.2

Generation time The time required to for a population to double (doubling time) in number. Ex. Escherichia coli (E. coli) double every 20 minutes Ex. Mycobacterium tuberculosis double every 12 to 24 hours 4-4

Principles of Bacterial Growth Growth can be calculated Nt = N0 x 2n (Nt ) number of cells in population (N0 ) original number of cells in the population (n) number of divisions Example N0 = 10 cells in original population n = 12 4 hours assuming 20 minute generation time Nt = 10 x 212 Nt = 10 x 4,096 Nt = 40,960

Growth in Batch Culture Bacteria growing in batch culture produce a growth curve with up to four distinct phases. Batch cultures are grown in tubes or flasks and are closed systems where no fresh nutrients are added or waste products removed. Lag phase occurs when bacteria are adjusting to them medium. For example, with a nutritionally poor medium, several anabolic pathways need to be turned on, resulting in a lag before active growth begins. In log or exponential phase, the cells are growing as fast as they can, limited only by growth conditions and genetic potential. During this phase, almost all cells are alive, they are most nearly identical, and they are most affected by outside influences like disinfectants. Due to nutrient depletion and/or accumulation of toxic end products, replication stops and cells enter a stationary phase where there is no net change in cell number. Death phase occurs when cells can no longer maintain viability and numbers decrease as a proportion.

Growth in Batch Culture

Mean Generation Time and Growth Rate 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 units of minutes. Growth rate (min-1) = Mean generation time can be determined directly from a semilog plot of bacterial concentration vs time after inoculation

Mean Generation Time and Growth Rate

Basic Chemostat System

Lab Chemostat System

Environmental factors Temperature Oxygen requirement pH Water availability 4-13

Temperature Enzymes, the machinery of the cell, are influenced by external factors and can be shown to have a range where they function that includes an optimal value that produces the highest activity. The range of enzyme activity determines the range for growth of specific bacteria, analogously leading to a value for optimal growth rate. In the case of temperature, bacteria are divided into categories based on the temperature range where they can grow and the temperature that provides optimal growth. 4-14

Temperature Psychrophile Psychrotroph Mesophile Thermophiles 0o to 18o C Psychrotroph 20°C to 30°C Important in food spoilage Mesophile 25°C to 45°C More common Disease causing Thermophiles 45°C to 70°C Common in hot springs and hot water heaters Hyperthermophiles 70°C to 110°C Live at very high temperatures, high enough where water threatens to become a gas Usually members of Archaea Found in hydrothermal vents

Oxygen requirements Oxygen is a very reactive molecule and can affect cells in several ways. The effect of oxygen is often determined using thioglycollate broth, a special medium that contains a reducing agent (thioglycollate) that removes oxygen so that a gradient occurs within the tube. Obligately aerobic bacteria can obtain energy only through aerobic respiration and have to have oxygen available. Thus, they will grow only at the surface of thioglycollate broth. Obligately anaerobic bacteria die in the presence of oxygen and can only grow at the bottom of thioglycollate broth. Some anaerobes are so sensitive to oxygen that even thioglycollate broth is not anoxic enough to provide suitable anaerobic conditions. Microaerophiles require oxygen for growth but the 20% in air is too toxic. As a result, they grow near the top but beneath the surface of thioglycollate broth where the oxygen concentration is typically 4 – 10%. Facultative anaerobes can use oxygen for aerobic respiration but can switch to fermentative metabolism in the absence of oxygen. As a result, they will grow throughout thioglycollate broth. (Heavier growth at top.) Aerotolerant anaerobes are anaerobic bacteria that can grow in the presence of air. (Not shown in diagram.) 4-16

pH Neutrophiles grow best around neutral pH (7) Acidophiles grow best at pH < 7 Alkophiles grow best at pH > 7 Acidotolerant grow best at pH 7 but can also grow at lower pH Alkotolerant grow best at pH 7 but can also grow at higher pH 4-17

Water Activity Liquid water is essential for life. Aqueous solutions actually have different amounts of water available, depending on how many solutes are dissolved in it. As a very simple model, consider two glasses, one full of pure water, the other containing the same amount of water plus a sponge. Which one would be easier to drink? On a much smaller scale, dissolved solutes act like a sponge, making less water available. Water activity (aw) can be decreased by the addition of any soluble molecule although salt (NaCl) and sugars are probably the most common.

Water Activity Microbes that require a high water activity (near or at 1) are termed nonhalophiles. (Halophile = salt-loving) Some bacteria require salt to grow and are called halophiles. If a very high concentration of salt is required (around saturation), the organisms are termed extreme halophiles. A nonhalophile that can grows best with almost no salt but can still grow with low levels of salt (~ 7%) is called halotolerant. In general, fungi are more tolerant of low water activity. (That’s why your jelly is more likely to get contaminated by fungi than bacteria.)

Nutritional Requirements Growth of prokaryotes depends on nutritional factors as well as physical environment Main factors to be considered are: Required elements Growth factors Energy sources Nutritional diversity

Nutritional Requirements Major elements (CHONPS + K, Mg, Fe, Ca) Carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, magnesium, iron, and calcium Essential components for macromolecules Organisms classified based on carbon usage Heterotrophs Use organism carbon as nutrient source Autotrophs Use inorganic carbon (CO2) as carbon source Trace elements (Co, Cu, Ni, Zn, Se, Mg, Wo) Cobalt, zinc, copper, molybdenum and manganese Required in minute amounts Assist in enzyme function Nutritional diversity Different organisms require the same nutrients but may require different forms of the nutrients

Major elements

Element % dry wgt Source Carbon 50 organic compounds or CO2 Oxygen 20 H2O, organic compounds, CO2, and O2 Nitrogen 14 NH3, NO3, organic compounds, N2 Hydrogen 8 H2O, organic compounds, H2 Phosphorus 3 inorganic phosphates (PO4) Sulfur 1 SO4, H2S, So, organic sulfur compounds Potassium Potassium salts Magnesium 0.5 Magnesium salts Calcium Calcium salts Iron 0.2 Iron salts

Carbon Source Organic molecules Inorganic carbon (CO2) Heterotrophs Autotrophs

Nitrogen Source Organic nitrogen Oxidized forms of inorganic nitrogen Primarily from the catabolism of amino acids Oxidized forms of inorganic nitrogen Nitrate (NO32-) and nitrite (NO2-) Reduced inorganic nitrogen Ammonium (NH4+) Dissolved nitrogen gas (N2) (Nitrogen fixation)

Phosphate Source Organic phosphate Inorganic phosphate (H2PO4- and HPO42-)

Sulfur Source Organic sulfur Oxidized inorganic sulfur Sulfate (SO42-) Reduced inorganic sulfur Sulfide (S2- or H2S) Elemental sulfur (So)

Growth Factors Some bacteria cannot synthesize some cell constituents These must be added to growth environment Referred to as growth factors Organisms can display wide variety of factor requirements Some need very few while others require many These termed fastidious Typical molecules Amino acids Nucleotide bases Enzymatic cofactors or “vitamins”

Culture Media Complex (contains undefined components) Chemically defined (all concentrations are known) Selective (favors the growth of a particular organism or group of organisms) Differential (has reactions that give isolates different appearance) Anaerobic (oxygen-free) 4-30

4-31

Characteristics of Media