Lecture 4 - Bacteria Nutrition, Metabolism And Growth

Bacteria nutrition, metabolism and growth. Dr. Edet E. Udo PhD Department of Microbiology Faculty of Medicine, Kuwait University.

• 1. Gram- positive bacteria contain a thick layer of peptidoglycan (T/F) • 2. The periplasmic space is present in both Gram-positive and Gramnegative bacteria ( T/F) • 3. Bacterial LPS has both antigenic and virulent properties (T/F) • 4. Staphylococcus is Gram-positive cocci in clusters (T/F) • 5. A bacterial spore germinates into two daughter cells ( T/ F). • • • • • •

Match the following 1. Gram- positive cell wall contains____________ 2. Gram positive cocci in chains __________________ 3. Has no cell wall ________________ 4. A primary pathogen __________________________ 5. An opportunistic pathogen _________________________

• E.coli, Klebsiella spp, Streptococcus, Haemophilus influenzae, Teichoic acid, Vibro cholerae, Mycoplasma,

Bacteria nutrition, metabolism and growth. • Objectives • Define nutrition, explain the nutritional requirements of bacteria. • Define bacterial growth. • Discuss the phases of microbial growth and their relationship to generation time • Define metabolism and the fundamental differences between anabolism and catabolism • Describe cultivation of bacteria in the laboratory

Bacterial nutrition • Definition: • Nutrition • Nutrition is a process by which organisms acquire chemical substances (Nutrients) used in cellular activities such as metabolism and growth. Organisms differ in the use of particular elements, their source and chemical form. Microbial growth Microbial growth refers to both the increase in cell size and number of cells in a population

Metabolism includes all the biochemical reactions that occur in the cell. It consists of anabolic and catabolic reactions. • Categories of essential nutrients • Macronutrients: required in relatively large quantities e.g. proteins, carbohydrates • Micronutrients or trace elements: required in smaller amounts. • E.g. zinc, manganese. • Are involved in enzyme functions and maintenance of protein structure.

Bacterial growth Cell division: – binary or transverse division.

• During binary fission, – the parent cell enlarges, duplicates its chromosomes and forms a central transverse septum that divides the cell into two daughter cells.

Cell division Generation or doubling time: The average generation time for bacteria is 30-60 minutes under optimum conditions. Most pathogens such as Staphylococcus aureus and Escherichia coli double in 20 – 30 minutes. The longest generation time requires days. E.g. Mycobacterium leprae that causes leprosy doubles in 20 to 30 days.

Planes of division

The growth curve

The growth curve • The growth curve is a graphic representation of closed population of bacteria overtime • This occurs in four phases, lag, log, stationary and decline phases.

• 1. The lag phase,

• Cells adjust to new environment. • There is no change in the number of cells but metabolic activity is high leading to increase in cellular components.

• 2. The log or exponential phase,

• Bacteria multiply at the fastest rate possible under the conditions provided. • Are susceptible to cell wall active antibiotics • Form metabolic end products

• 3. The stationary phase • there is an equilibrium between cell division and cell death caused by : • decrease in nutrient, • increase in cell population and accumulation of metabolic waste / end products e.g antibiotics. • Sporulating cells initiate spore formation

• 4. Death or Decline phase

•

The number of death cells exceeds the number of new cells formed due to lack of nutrients and accumulation of toxic waste

•

Factors affecting bacterial growth Nutritional requirements

• Chemical: water, carbon source, nitrogen, minerals, oxygen, growth factors

• Carbon Source: • All bacteria require carbon for growth. • Bacteria can been classified on the basis of their carbon source. – Autotrophs: use carbon dioxide as the sole source of carbon – Heterotrophs: use more complex organic compounds such as carbohydrates and amino acids as source of carbon – Photoautotroph- energy need is supplied by light – Chemoautotroph - energy is extracted from inorganic substances

• Inorganic nutrients (ions): – Contain no carbon and hydrogen atoms : phosphates, potassium, magnesium, nitrogen, sulfur, iron and numerous trace metals. • Organic nutrients: • contain carbon and hydrogen atoms. Include carbohydrates, lipids amino acids, Nucleic acids etc. • Carbohydrates: • are used as the initial carbon source for many biosynthetic pathways and as electron donors (energy source) by many bacteria. • Amino acids • are important source of carbon and nitrogen.

Nutritional factors • Phosphorus • is present as phosphates salts. • They function in energy metabolism and as constituents of nucleic acids, phospholipids, teichoic acids, ATP, etc • Minerals: • K, Mg, Ca, Fe are required in relatively high levels. • Function as cofactors in enzyme reactions and as cations they act as buffers within the cells • Vitamins, • purines and pyrimidines ( accessory growth factors) function as coenzymes

Physical factors • Oxygen requirement: • Bacteria can be divided into five groups on the basis of their oxygen requirements • 1. Obligate or strict aerobes: • The growth of bacteria is inhibited by absence of oxygen An example of a strict or obligate aerobe is Pseudomonas aeruginosa • 2. Obligate anaerobes: • Growth is inhibited by the presence of oxygen. – Examples of obligate anaerobe are Clostridium spp and Bacteriodes spp.

• 3. Facultative anaerobes • are able to grow in the presence or absence of molecular oxygen. E.g. include staphylococci, streptococci, Enteococci, etc. • 4. Microaerophilic bacteria: • grow best under increased carbon dioxide tension. Examples include Neisseria gonorrhoeae, Haemophilus influenzae • 5. Aerotolerant bacteria: • can survive (but not grow) for a short period of time in the presence of atmospheric oxygen • Tolerance to oxygen is related to the ability of the bacterium to detoxify superoxide and hydrogen peroxide produced as bye products of aerobic respiration.

• 1. Superoxide dismutase, • which converts superoxide ( a toxic metabolite) into hydrogen peroxide is present in aerobic and aerotolerant bacteria but not in obligate anaerobes. • 2. Catalase, • which converts hydrogen peroxide into water and oxygen is also present in all aerobic bacteria but is lacking in aerotolerant organisms. Strict anaerobes lack both enzymes. • O2- + O2- -superoxide dismutase H2O2 (Hydrogen peroxide) + O2• H2O2 + H2O2 Catalase-------2H2O +O2

• Temperature: • There are three critical temperature ranges for growth: • (a) Minimum temperature• (b). Maximum temperature: • ©. Optimum temperature: • Psychrophiles: • Has optimum temperature below 15 C but capable of growth at 0 C • Mesophiles: – grow at a range of 20 –40 C. Include most bacterial pathogens with optimum temp. at 37 C • Thermophiles: – microbes that has optimum temperature above 45 C with a general range of 45-80 C • Most thermophiles form spores e.g. Bacillus steareothermophilus

• pH: • Optimum pH for most bacteria is near pH 7.0 (pH 6.5- pH 7.5) • Bacteria can be classified as alkalinophiles, neutrophiles or acidophiles according to their degree of tolerance to pH changes

Osmotic pressure: • When a microbial cell is in a hypertonic solution cellular water moves out of the cell through the cell membrane to the hypertonic solution. • This osmotic loss of water causes shrinkage of the cell PLASMOLYSIS • In a hypotonic solution such as in distilled water, water will enter the cell and the cell may be lysed by such treatment (PLASMOPTYSIS). • Halophiles • require high salt concentrations for growth. Some bacteria can tolerate 15% salt. E.g. S. aureus

Bacterial metabolism • Metabolism consists of catabolic and anabolic reactions. – Anabolic reactions are energy-requiring subset of metabolic reactions, which synthesize large molecules from smaller ones. – Catabolic reactions are the energy – releasing subset of metabolic reactions, which degrade or breakdown large molecules into smaller ones. • Metabolism is best considered in three stages – 1. Energy metabolism – 2. Respiratory metabolism

• 1. Energy metabolism – Energy used by bacteria is primarily produced by fermentative and/ or respiratory metabolic pathways. • Metabolic reactions are catalyzed by enzymes – Anaerobic metabolism (fermentative metabolism – A. Glycolytic ( Embden-Meyerhof ) pathway – The major glucose utilization pathway. – Glycolysis- the metabolism of glucose to yield pyruvic acid and 2 ATP molecules – B. Entner-Doudoroff pathway • Aerobic metabolism ( respiration) • The Krebs cycle • Involves the the metabolism of 2-carbon

• Biosynthetic pathways – Used to build small molecules with nitrogen, sulphur and other minerals into amino acids, purines, pyrimidines, polysaccharides and lipids

Bacterial metabolism • Uses of energy • For biosynthetic activities e.g. cell wall synthesis, protein synthesis, nucleic acid synthesis etc • Membrane transport and movement e.g active transportation. • Uses of metabolic products in the laboratory • Metabolic end products e.g. pyruvic acid, lactic acid, mixed gases, alcohols, are used for bacterial identification. (Biochemical tests) e.g. oxidase tests, catalase tests.

Cultivation of bacteria in the laboratory • Culture media: • Any material prepared for the growth of bacteria in the laboratory. • They can be broth (liquid) or • solid media ( contains a gelling substance such as agar) • Microbes that grow on a culture medium are known as a culture. • When grown on solid media, the growth is called colony . • Growth in a liquid medium is demonstrated by turbidity.

Types of Culture media: • Defined synthetic medium: – contains known quantities of specific nutrients – contain nutrients of reasonably well– Complex medium: – known composition that varies from batch to batch • Enrichment medium: (liquid medium) – is used to encourage the growth of a particular organism in a mixed culture • Enriched media: (Solid medium,) – contain additional nutrients to support the growth of fastidious organisms. E.g blood agar and chocolate agar

• Chocolate agar contains lysed blood. • The lysis releases intracellular nutrients such as haemin (X factor) and the coenzyme nicotinamide adenine dinucleotide (NAD or V factor) into the agar for use by fastidious microorganisms such as Haemophilus species and Neisseria gonorrhoeae.

• Selective media: •

contains salts, dyes or other chemicals that inhibit the growth unwanted microorganisms.

• Differential media: •

contain chemicals that allow the distinction between different types of organisms e.g. Lactose in MacConkey agar.

• Preservation of bacterial cultures: •

By refrigeration,, lyophilization, storage in liquid nitrogen

Culture media