Environmental Influences and Control of Microbial Growth Chapter 5 Environmental Influences and Control of Microbial Growth

Chapter Overview - Physical, chemical, and biological ● How the environment limits growth ● The microbial response to temperature ● How microbes cope with pressure ● The microbial response to changes in: water activity, salt concentrations, pH, and oxygen ● Hungry microbes ● The control of microbes: - Physical, chemical, and biological

Introduction Microbes have both the fastest and the slowest growth rates of known organisms. Some hot-springs bacteria can double in as little as 10 minutes, whereas deep-seas sediment microbes may take as long as 100 years. These differences are determined by nutrition and niche-specific physical parameters like temperature and pH.

Environmental Influences and Control of Microbial Growth Lactobacillus plantarum pH 6.5 pH 3

Environmental Limits of Microbial Growth “Normal” growth conditions - Sea-level; temperature 20–40oC; neutral pH; 0.9% salt; and ample nutrients Any ecological niche outside this window is called “extreme,” and organisms inhabiting them are called extremophiles. Figure 1.1

Genomic and Proteomic approaches to study gene expressions Global approaches used to study gene expression allow us to view how organisms respond to changes in their environment. - DNA microarrays assess which RNAs (Transcripts) are made in a given organism under a given condition. - Two-dimensional protein gels achieve separation of proteins based on differences in each protein’s isoelectric point (first dimension) and molecular weight (second dimension). 6

The 2-D gel image shows protein expressed under 2 pH conditions Response to environmental stress: Global analysis: Genes and proteins expressed under two different growth conditions. The microarrays contain all genes on a genome attached to a slide. Organisms grown under two growth conditions. Transcripts (RNAs) are converted to cDNA and hybridized to the slide. Red indicate gene expressed under one conditions and Green under different conditions. Yellow shows expression under both conditions. The 2-D gel image shows protein expressed under 2 pH conditions 7

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Effect of Temperature Changes in temperature impact every aspect of microbial physiology. Each organism has an “optimum, minimum and maximum” temperatures that define its growth limits. These are cardinal temperatures

Temperature Optimum Microorganisms can be classified by their growth temperature: - Psychrophiles ~ 0–20oC (15oC) - Mesophiles ~ 15–45oC (37 oC) - Thermophiles ~ 40–80oC (60 oC) - Hyperthermophiles ~ 65–121oC (90oC) 10

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Bacterium from South Polar snow Psychrophiles can be found in Icebergs Methanocaldococcus jannaschii, 780 C and 30 psi. Isolated from 2600 m deep “white smoker” chimney Yellowstone hotspring Thermus aquatics, Yellowstone 12

How do microbes deal with cold? Cytoplasmic membrane contain high-level of unsaturated fatty acids allowing their membranes to be more flexible in cold. Contain antifreeze proteins and cryoprotectants such as trehalose Enzymes from psychrophiles are useful for biotechnology such a bioremediation and for biochemical reactions at low temperature Trehalose

Cytoplasmic membrane contain high-level of unsaturated fatty acids allowing their membranes to be more flexible in cold.

How do microbes deal with heat-Shock? Rapid temperature changes experienced by microbes show the expression of certain genes. The proteins, chaperones help maintain protein shape (3-D structure). Specialized DNA binding proteins protect DNA from denaturation. Membranes have high levels of saturated fatty acids or hydrocarbons. Some archaea have single lipid layer in membrane as opposed to bilayer 15

Archaeal Lipids Extreme temperature archaea

Variations in Pressure Barophiles or piezophiles are organisms adapted to grow at very high pressures. - Up to 1,000 atm (101 MPa, or 14,000 psi) Barotolerant organisms grow well over the range of 1–50 MPa, but their growth falls off thereafter. Note that many barophiles are also psychrophiles because the average temperature at the ocean floor is 2oC. 17

Figure 5.5 Figure 5.6 18

Changes in Water Activity Water activity (aw) is a measure of how much water is available for use. Osmolarity is a measure of the number of solute molecules in a solution and is inversely related to aw. Aquaporins are membrane-channel proteins that allow water to traverse the membrane much faster than by diffusion. - Help protect the cell from osmotic stress

Minimizing Osmotic Stress In addition to moving water, microbes have at least two mechanisms to minimize osmotic stress: - In hypertonic media, bacteria protect their internal water by synthesizing or importing compatible solutes (e.g.: proline or K+) - In hypotonic media, pressure-sensitive or mechanosensitive channels can be used to leak solutes out of the cell. 20

aw = Vapor Pressure of a Solution Water Activity (aw) aw = Vapor Pressure of a Solution Vapor Pressure of Pure water aw of solutions ranges from 0-1. aw of pure water is 1 (100% humidity).

Changes in Salt Concentrations Halophiles require high salt concentrations. - From 2–4 M NaCl (10–20% NaCl) - For comparison, seawater is ~ 3.5% NaCl 22

Changes in pH Figure 5.11 23

Changes in pH All enzyme activities exhibit optima, minima, and maxima with regard to pH. Bacteria regulate internal pH. - When environment is in a similar pH range Weak acids can pass through membranes. - Disrupt cell pH homeostasis, and thus will kill cells - This phenomenon is used to preserve foods. 24

Changes in pH Three classes of organisms are differentiated by the pH of their growth range: - Neutralophiles grow at pH 5–8. - Include most pathogens - Acidophiles grow at pH 0–5. - Are often chemoautotrophs - Alkaliphiles grow at pH 9–11. - Typically found in soda lakes 25

- It is also a major food for the famous pink flamingo. The cyanobacterium Spirulina has high concentrations of carotene, giving it a distinct pink color. - It is also a major food for the famous pink flamingo. Figure 5.15 26

pH Homeostasis When cells are placed in low pH conditions, protons can enter the cell and lower internal pH to lethal levels. Microbes can prevent the unwanted influx of protons by exchanging extracellular K+ for intracellular H+ when the internal pH becomes too low. Under extremely alkaline conditions, the cells can use the Na+/H+ antiporter to bring protons into the cell in exchange for expelling Na+. 27

Figure 5.17 28

Microbial Responses to Oxygen Strict aerobes can only grow in oxygen. Microaerophiles grow only at lower O2 levels. Strict anaerobes die in the least bit of oxygen. Facultative anaerobes can live with or without oxygen. Aerotolerant anaerobes grow in oxygen while retaining a fermentation-based metabolism.

Oxygen-related growth zones in a standing test tube Figure 5.19 30

Generation and destruction of reactive oxygen species (ROS) Figure 5.20 31

Culturing Anaerobes in the Lab Three oxygen-removing techniques are used today: 1. Special reducing agents (thioglycolate) or enzyme systems (Oxyrase) can be added to ordinary liquid media. 2. An anaerobe jar 3. An anaerobic chamber with glove ports - O2 is removed by vacuum and replaced with N2 and CO2. 32

Figure 5.21 33

Microbial Response to Starvation Starvation is a stress that can elicit a “starvation response” in many microbes. - Enzymes are produced to increase the efficiency of nutrient gathering and to protect cell macromolecules from damage. This response is usually triggered by the accumulation of small signal molecules such as cAMP or guanosine tetraphosphate. 34

Microbial Response to Starvation Some organisms growing on nutrient-limited agar can even form colonies with intricate geometrical shapes that help the population cope, in some unknown way, to food stress. Figure 5.22 35

Oligotrophic Bacteria In natural ecosystems, most microbes appear to be oligotrophs, organisms with a high rate of growth at low solute concentrations. - Indeed, they require low nutrient levels to survive. Some oligotrophic bacteria have thin extensions of their membrane and cell wall called prothecae (stalks). - These expand the surface area of the cell and increase nutrient-transport capacity. 36

Humans Influence Microbial Ecosystems Maximum diversity in an ecosystem is maintained, in part, by the different nutrient-gathering profiles of competing microbes. Figure 5.23 37

Humans Influence Microbial Ecosystems Eutrophication is the sudden infusion of large quantities of a formerly limiting nutrient. - It can lead to a “bloom” of microbes, which can threaten the existence of competing species. Figure 5.24 38

Control of Microbes A variety of terms are used to describe antimicrobial control measures: - Sterilization: Killing of all living organisms - Disinfection: Killing or removal of pathogens from inanimate objects - Antisepsis: Killing or removal of pathogens from the surface of living tissues - Sanitation: Reducing the microbial population to safe levels 39

Microbes die at a logarithmic rate. Decimal reduction time (D value) is the length of time it takes an agent or a condition to kill 90% of the population. Figure 5.25 40

Physical Agents High temperature - Moist heat is more effective than dry heat. - Boiling water (100oC) kills most cells. - Killing spores and thermophiles usually requires a combination of high pressure and temperature. - Steam autoclave - 121oC at 15 psi for 20 minutes 41

Figure 5.26 42

Physical Agents Pasteurization - Many different time and temperature combinations can be used. - LTLT (low-temperature/long-time) - 63oC for 30 minutes - HTST (high-temperature/short-time) - 72oC for 15 seconds - Both processes kill Coxiella burnetii, the causative agent of Q fever. 43

Physical Agents Cold - Low temperatures slow down growth and preserve strains. - Refrigeration temperatures (4–8oC) are used for food preservation. - For long-term storage of cultures - Placing solutions in glycerol at –70oC - Lyophilization or freeze-drying 44

Physical Agents Filtration - Micropore filters with pore sizes of 0.2 mm can remove microbial cells, but not viruses, from solutions. Figure 5.27 45

Air can also be sterilized by filtration. Laminar flow biological safety cabinets force air through HEPA filters. Figure 5.28 46

Physical Agents Irradiation - Ultraviolet light - Has poor penetrating power - Used only for surface sterilization - Gamma rays, electron beams, and X-rays - Have high penetrating power - Used to irradiate foods and other heat- sensitive items 47

Chemical Agents A number of factors influence the efficacy of a given chemical agent, including: - The presence of organic matter - The kinds of organisms present - Corrosiveness - Stability, odor, and surface tension 48

The Phenol Coefficient The phenol coefficient test compares the effectiveness of disinfectants. Table 5.3 49

Commercial Disinfectants and Antiseptics These include: - Ethanol - Iodine (Wescodyne and Betadine) - Chlorine All of the above damage proteins, lipids, and DNA. - Are used to reduce or eliminate microbial content from objects 50

Figure 5.30 51

Antibiotics Antibiotics are chemical compounds synthesized by one microbe that kill or inhibit the growth of other microbial species. Penicillin mimics part of the bacterial cell wall. - Prevents cell wall formation and is bactericidal Figure 5.31 52

Effect of ampicillin (a penicillin derivative) on E. coli Figure 5.32 53

Biological Control of Microbes Biocontrol is the use of one microbe to control the growth of another. - Probiotics contain certain microbes that, when ingested, aim to restore balance to intestinal flora. - Lactobacillus and Bifidobacterium - Phage therapy aims to treat infectious diseases with a virus targeted to the pathogen. - A possible alternative to antibiotics in the face of rising antibiotic resistance 54

Chapter Summary - Psychrophiles, mesophiles, and thermophiles ● Global analysis of genes and proteins allows us to study how microbes react to environmental changes. ● Microbes are classified by growth temperature: - Psychrophiles, mesophiles, and thermophiles ● Barophiles can grow at very high pressures. ● Halophiles require high salt concentrations. ● Microbes are classified by pH range: - Acidophiles, neutralophiles, and alkaliphiles ● Microbes are classified by their O2 requirements: - Aerobes, facultative, microaerophiles, and anaerobes 55

Chapter Summary ● Cells treated with antimicrobials die at a logarithmic rate. ● Physical agents used to control microbes include: - Autoclaving, pasteurization, refrigeration, filtration, and irradiation ● Chemical agents used to control microbes include: - Antiseptics and disinfectants ● Antibiotics selectively control microbial growth. ● Biological control of microbes includes the use of probiotics and phage therapy. 56