BACTERIAL METABOLISM AND GROWTH

DR A.R. OJEWUYI

Introduction

• Organisms use a variety of nutrients for:

- Their energy needs

- Building organic molecules & cellular structures

Introduction

• All bacteria have three major nutritional needs for growth:

- A source of carbon: for making cellular constituents, carbon represents 50% of the dry weight of the bacterium.

- A source of Nitrogen: for making proteins.

Nitrogen makes up 14% of the dry weight

- A source of energy: ATP, for performing cellular functions.

Introduction

• Other molecules needed are :

- Phosphate for nucleic acids, phospholipids of cell membranes and sulphur for protein synthesis.

- A variety of metals and ions such as Calcium, Copper, Iron, Magnesium, Manganese, Phosphorus, Sodium and Potassium are also needed for enzymatic activity.

- Trace elements are needed in extremely small amounts, can be obtained through water intake.

Nutritional Requirements for Growth

Bacteria are classified into two basic groups according to how they meet their nutritional needs

• Autotroph: use CO2 as the sole source of carbon, with only H2O and inorganic salts required in addition

•Chemoautotroph: unique metabolism, use chemical energy from inorganic molecules, Sulfur and Iron

•Phototrophs: obtains energy photosynthetically

Nutritional Requirements for Growth

• Heterotrophs require more complex substances for growth. They require an organic source of carbon, such as glucose, and obtain energy by oxidizing or fermenting organic substances.

• All bacteria that inhabit the human body fall into the heterotropic group.

Microbial Growth is affected by two major factors:

1.Environmental: Temperature, pH and gaseous composition of the atmosphere

2.Chemical: Proper concentrations of C, H, O, N, P, S, some trace elements, and some organic cofactors

Environmental Factors Influencing Growth

Physical factors that affect bacterial growth; Temperature

• Mesophiles : grow best moderate temp. 25°C – 40°C. Most bacteria that have adapted to humans are mesophiles that grow best near human body temp. (37°C)

• Psychrophiles: adapted to survive and grow at cooler temp., even in the frigde (10°C-20°C)

• Thermophiles: adapted to and grow at much higher temp.(50°C-60°C)

Physical factors that affect bacterial growth: pH

• Bacteria grow best at pH range of 6.5 to 7.5

• Halophilic Vibrio spp

• Helicobacter pylori can survive in acidic conditions.

Atmospheric Requirement

• Obligate aerobes: require molecular oxygen (as final electron acceptor in catabolism)

• Obligate anaerobes: require atmosphere with no O2,

an organic molecule is final electron acceptor in catabolism (like a fermentation pathway)

• Facultative anaerobes: grow with or without O2, usually are also fermenters.

• Capnophilic: grow best in higher carbon dioxide concentration (5% to 10%).

Using oxygen in metabolism creates toxic waste.

Microbes that are able to use aerobic respiration produce enzymes to detoxify oxygen:

Catalase: H2O2 --- H20 and 02

Superoxide dismutase: O2⁻ + O2 - + 2H⁺ --- H2O2 + O2

Microbes that don’t make these enzymes cannot exist in the presence of oxygen.

Microbes & Oxygen

Bacteria Growth

• Bacteria reproduce by “binary fission”, a cell divides into two, two to four, four to eight, etc.

• Cell division can occur quite rapidly depending on nutrient levels, temperature, etc. E. coli candivide every 20 minutes (time to double –generation time) in nutrient media at 37°C.

• The numbers get so large we express them as the log of the number of cells.

Bacteria Growth Curve

A= Lag phase

B= Log phase

C= Stationary phase

D= Death Phase

Generation Time Under Optimal Conditions (at 37oC)

Organism Generation Time

Bacillus cereus 28 min

Escherichia coli 12.5 – 20 min

Staphylococcus aureus 27-30 min

Mycobacterium tuberculosis 18 – 24 hrs

Treponema pallidum 30 hrs

How do we measure growth of bacteria in a growth curve?

•Direct cell count using stained slides that have a grid for counting.

•Indirect: Serial dilution, plates are inoculated - incubated and colonies counted. Number of colonies X dilution factor gives the number of bacteria.

•Density measurement: The cloudiness or turbidity of a bacteria broth culture in a log phase can be correlated to the CFU/ml.

Bacteria Metabolism

• Microbial metabolism consists of the biochemical reactions bacteria use to break down organic compounds and those used in synthesis of new bacteria parts from the resulting carbon skeleton

• Energy for new construction is generated during the metabolic breakdown of the substrate

• Metabolic regulation can be through regulation of involved enzymes or their products

Bacteria Metabolism

Metabolism involves

- Catabolism

- Anabolism

• Both occur simultaneously in cells

• Catabolism eventually produces the chemical energy (ATP) required for all cellular functions such as anabolism (synthesis), membrane transport, etc.

• Bacteria vary in their ability to use various compounds as substrates and in the end products generated.

• The metabolic end products generated are used phenotypically in identification of these organisms.

• There is a variety of biochemical pathways for substrate breakdown, the particular partway used determines the end product and final pH of the medium.

Fermentation and Respiration

• The two mechanisms employed in catabolism of carbohydrates by bacteria are:

- Fermentation

- Respiration (oxidation)

• Fermentation is an anaerobic process carried out by both obligate and facultative anaerobe

• In fermentation, the electron acceptor is an organic compound, but its less efficient in energy generation than respiration(oxidation)

• Such end-products as lactate, butyrate, ethanol and acetoin can accumulate in the medium after fermentation

Respiration(oxidation)

• It is not an act of breathing, but an efficient energy –generating process in which molecular oxygen is the final electron acceptor.

• Aerobic respiration is seen in obligate aerobes and facultative anaerobes with oxygen being the final electron acceptor.

• Certain anaerobes can carry out anaerobic respiration using inorganic forms of oxygen like nitrate and sulphate as the final electron acceptor.

Biochemical Partways from Glucose to Pyruvic Acid

• The starting carbohydrate for bacteria fermentations or oxidations is glucose

• When bacteria used other sugars as a carbon, they first convert the sugar to glucose which is then processed by one of the three partways :

- Embden-Meyerhof-Parnas (EMB) Glycolytic Pathway

- Pentose Phosphate (Phosphogluconate) Pathway

- Entner- Doudoroff Pathway

Oxidation – Reduction•Organic molecules like glucose have covalent bonds between C-C, C-H, C-O,O-H C6H12O6

•When the molecule is broken down -, the covalent bonds are broken –electrons are removed and transferred to carrier molecules.

•Oxidation is the removal of electrons and/or adding Oxygen

•In Glycolysis the glucose is broken into two Pyruvates,

•The electrons and a H+ are transferred to a carrier, NAD.

•NAD gains the electron (and Hydrogen too), it is reduced

to NADH, thus oxidation and reduction go together.

Oxidation – Reduction

•Look again at glycolysis.

•Glucose is oxidized and the carrier NAD is reduced.

•For every glucose, two Carriers are produced

2 NADH (what happens to them, they have to

be regenerated – oxidized back to NAD)

•Aerobes eventually produce CO2 and H2O

•Thus oxygen is the final electron acceptor( producing

Water).

•Anaerobes use a different set of enzymes, a

Fermentative pathway that generates other acids,

alcohols or gasses (lactic acid, ethanol, CO2)

** electron acceptor is an “organic molecule”**

•If no regeneration of NAD, the glycolysis pathway

shuts down and the organism dies – no ATP

•Glycolysis, no oxygen, fermentation, only 2 ATP per molecule of glucose

•Glycolysis, with oxygen, followed by Krebs and electron transport, can

generate much more ATP (sometimes as much as 36).

• Aerobic mechanisms are much more energy efficient.

•In the Krebs cycle many more carrier molecules like NADH are generated

and thus lead to more ATP. (Other carriers FAD, NADP – we just use

NAD as a representative type of carrier).

•The constantly turning of the cycle produces a steady stream of reduced

carriers (NADH) which pass the electrons to a set of carrier-processor

molecules imbedded in the membrane of the Mitochondria. These

carriers are called the “electron” transport chain.

Assignment

Have a glance at the following pathways and

note the differences.

- Embden-Meyerhof-Parnas (EMB) Glycolytic Pathway

- Pentose Phosphate (Phosphogluconate) Pathway

- Entner- Doudoroff Pathway

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