Chapter 7

Microbial Nutrition, Ecology, and Growth

Microbial Nutrition

•          Nutrition is a process of acquiring chemical substances from the environment

•          The absorbed nutrients are used

–        for energy yielding processes

–        growth

•          The chemical elements absolutely needed - essential nutrients

–        Macronutrients: C, H, O…

–        Micronutrients: Mn, Zn, Ni…

Sources of Essential Nutrients

Carbon

•          Structural backbone of living matter

–        50% of microbial dry weight is C

–        Autotrophs derive C from CO2

–        Heterotrophs derive C from organic matter

Nitrogen

•          14% of microbial dry weight is N

–        required for protein, DNA, RNA, ATP synthesis

•          Microorganisms derive N by:

–        Breaking down proteins into amino acids (reuse of amino acids)

–        NH4, – ammonium ions

–        NO3 – nitrate

–        N2 – nitrogen fixers

•          Free-living

•          Symbionts with plants

Other Elements

•          Sulfur - synthesis of sulfur-containing amino acids

•          Phosphorus - synthesis of DNA, RNA, ATP and phospholipids of cell membrane

•          Trace elements – minerals needed as enzyme cofactors

•          Growth factors – organic chemicals that cannot be synthesized by certain organisms (vitamins, certain amino acids…)

Nutritional Types

 Heterotrophs

•          Chemoheterotrophs

–        Energy and Carbon source from organic molecules

•          Saprobes derive nutrients from dead organic material

–        Opportunistic pathogene – a saprobe infecting  the compromised host

•          Parasites derive nutrients from living organisms

–        Pathogenes – harm the host (Streptococcus)

–        Obligate intracellular parasites (Rickettsias, Chlamydias, Viruses)

 How microbes eat?

•          Absorb nutrients that are dissolved

•          The molecules need to be small

•          The big molecules are degraded by extracellular enzymes

•          Diffusion of water molecules through a selectively permeable membrane

•          Water molecules will move from the side that has more water to the side with less water

•          Until equilibrium is reached

 Osmotic variations

•          Depending on the concentration of water and solutes on either side of cell membrane, the cell can be subjected to: isotonic, hypotonic, and hypertonic osmotic conditions

•          Isotonic – water concentration is equal inside and outside

•          Hypotonic solutions have lower solute concentrations; cells placed in these solutions will swell and burst

•          In hypertonic solution the cellular water passes out of the cell -  Plasmolysis – shrinking of the cell content inside the plasma membrane

•          Used in food preservation.

•          High concentration of salt or sugar draws the water out of microbial cell.

 The Movement of molecules across the cell membranes

•          Diffusion – movement of molecules from an area of higher concentration to the area of lower concentration

•          Used for transport of small molecules (O₂)

 Facilitated diffusion

•          Substance to be transported -combines with the plasma membrane protein – transporter

•          This changes the shape of the transporter – substance is moved across the membrane and released

•          No energy needed

Active transport

•          Brings in molecules against a gradient

•          Involves

–        Membrane proteins – permeases

–        Pumps (transport of H⁺, K⁺, Na⁺)

•          Expenditure of energy (ATP)

•          Group translocation ( a type of active transport)

–        the substance is chemically altered while being transported into the cell

 Eating and drinking by eukaryotic cells

Endocytosis

•          Engulfing particles and molecules from the outside with the cell membrane

Pinocytosis

•          Absorbing liquids (oils)

Phagocytosis

•          White blood cells can ingest whole cells - bacteria

 Environmental Factors that Influence Microbes

•          Physical

–        Temperature    

–        pH

–        Osmotic pressure

•          Chemical

–        Elements (C, N,S,P)

–        Trace elements

–        Oxygen

–        Growth factors

 Temperature

•          Microorganisms have minimum, optimum and maximum growth temperatures

Categories of Microbes Based on Temperature Range

Ranges                 Optimum

•          Psychrophiles                  -10⁰ to 20⁰C         12⁰C

•          Psychotrophs                       0⁰ to 30⁰C          22⁰C

•          Mesophiles                          10⁰ to 50⁰C        37⁰C

•          Thermophiles                     40⁰ to 70⁰C        62⁰C

•          Hyperthermophiles         65⁰ to 110⁰C      94⁰0C

 Is it possible to make money on unusual microbes?

•          Thomas Brock (1965) isolated the bacterium Thermus aquaticus from the hot springs in  Yellowstone Park.

•          An unusual microorganism that grows at high temperatures

•          Later on, it was discovered that this organism possess an enzyme (DNA polymerase) involved in DNA synthesis that is active at 72⁰C

•          With the development of the PCR technology production of this enzyme became a multimillion-dollar business

About the hot springs

•          Hot spring – a natural discharge of groundwater with elevated temperatures

•          Geothermal energy- an alternative source of energy

•          Used for different purposes:

•          Bathing, heating, generating electrical energy

•          Hot springs are found all over the world

•          Volcanic areas

Oxygen requirement

•          Obligate aerobs – require O₂ to live

•          Facultative anaerobs – can grow in absence of O₂

•          Obligate anaerobs – killed by O₂

•          Microearophiles – require O₂ at concentrations lower than those in air

Oxygen is deadly for obligate anaerobes

•          How can this be true?

•          Superoxide free radical – O₂^- and H₂O₂ is formed during the normal metabolism

•          Aerobes produce superoxide dismutase to detoxify O₂^-  by reducing it to H₂O₂

                2H₂O₂                              2H₂O + O₂ 

Anaerobes lack superoxide dismutase

 Effect of pH

•          Most bacteria grow in pH range 6.5-7.5

•          Low and high pH inhibits growth of bacteria

•          Mild acids can help preserve foods by preventing further microbial growth (sauerkraut, pickles)

•          Acidophiles tolerant to acidity (bacteria and fungi)

•          Alkalinophiles live in alkaline soils and water up to pH 11.5

 Effect of Osmotic Pressure

•          Microorganisms that live in high salt concentration are called Halophyles

•          Obligate halophyles – require high salt concentrations to survive, grow in up to 30% salt

•          Facultative halophyles - tolerate high salt concentrations (up to 2%)

 Microbial Growth

•          There are two aspects of microbial growth:

•          Increase in the cell size

•          Increase in the cell number –The growth of bacterial culture

The growth of bacterial culture

•          Bacterial culture grows by doubling of individual cells (binary fission)

•          Growth of bacterial culture is defined by  “generation time”

•          Generation time (G.T.) is the time required for a cell to divide.

•          For most bacteria G.T. is  30 – 60 min 

•          E. coli -  20 min (1 cell after 20 generations will produce 1 million cells)

•          Logarithmic graphing is used to describe the growth of bacterial culture

Stages in the Normal Growth Curve

•          Lag phase – little or no change in cell number. Cells are metabolically active (enzyme, DNA  synthesis)

•          Log phase – exponential growth phase – active reproduction, high metabolic activity

•          Stationary phase - Number of microbial deaths = number of new cells. Metabolic activity slow.

•          Death phase The number of deaths greater than number of new cells

 Measurement of microbial growth

•          Direct Methods

–        Viable Plate Counts

–        Direct Microscopic Count

•          Indirect Methods

–        Turbidity (not covered in this lecture)

–        Metabolic activity (not covered in this lecture)

Plate Count

•          Suspension of cells (water, milk, urine) is inoculated onto agarized medium

•          Usually serial dilutions are required

•          One single cell is transformed into a visible single colony          

•          Only viable cells are detected

•          Plates that have between  30-300 colonies are counted

 Direct Microscopic Count

•          Specially designed slide - cytometer

•          Slide contains a well with inscribed squares of known area and volume

•          The cells are counted under the microscope, multiplied with the factor that gives the count per ml

•          Disadvantages of the method:

•          All cells are counted - including dead cells

•          Motile cells are difficult to count

•          High concentrations of the cell are required

•          Advantage of the method

•          It is fast - no need for incubation