microbial nutrition

1. MICROBIAL NUTRITION, GROWTH AND KINETICS

2. Nutrient requirements
• Nutrients: Substances in the environment used by the organisms for
catabolism and anabolism
• Microorganisms require about ten elements in large quantities
(Macronutrients).
• These include; carbon, hydrogen, oxygen, nitrogen, Sulphur,
phosphorus
• Are used to construct carbohydrates, lipids, proteins, and nucleic
acids.
• Potassium, calcium, magnesium and iron are needed as cations and
are part of enzymes and cofactors

3. Nutrient requirements
• Several other elements are needed in very small amounts and are
parts of enzymes and cofactors (Micronutrients).
• These include; iron, copper, molybdenum and zinc; referred as Trace
elements.
• Most are essential for activity of certain enzymes as cofactors.
• Contaminants in water, glassware, and regular media components
often are adequate for growth.

4. Nutrient requirements
GROWTH FACTORS
• Organic compounds, required in very small amount and then only
by some cells
• Are amino acids, purines, pyrimidines, and vitamins
• Amino acids are needed for protein synthesis
• Purines and pyrimidines are nucleic acid synthesis
• Vitamins are small organic molecules usually make up all or part of
enzyme co-factors
• Are required in very small amounts for growth

5. Nutritional types of microorganisms
• Organisms are classified according to the source of energy they use
• Heterotrophs; use carbon source as organic material, CHO
• Autotrophs; use carbon source as carbon dioxide, CO2
• Omnivores: use over 100 different carbon compounds
• Fastidious: catabolize only a few carbon compound
• Relatively indigestible human-made substances are metabolized by complex
populations of microorganisms

6. Nutritional types of microorganisms
• Phototrophs: use light as energy source.
• Chemotrophs: obtain energy from the oxidation of chemical
compounds.
• Lithotrophs: use reduced inorganic substances as their electron
source.
• Organotrophs: extract electrons from organic compounds.

7. Nutritional types of microorganisms

8. Mixotrophic
• Bacteria that rely on inorganic energy sources and organic (or
sometimes CO2) carbon sources.
• Include; many purple non sulfur bacteria, Beggiatoa
• They combine chemolithoautotrophic and heterotrophic metabolic
processes.
• No oxygen: photoorganotrophic heterotrophs
• Normal oxygen: oxidize organic molecules and function
chemotrophically
• Low oxygen: photosynthesis and oxidative metabolism

9. MICROBIAL GROWTH AND KINETICS
• Microbes grow via binary fission, resulting in exponential increases in
numbers
• The number of cell arising from a single cell is 2n after n generations
• Generation time is the time it takes for a single cell to grow and divide

10. MICROBIAL GROWTH AND KINETICS

11. The Rate of Population Growth
• Generation or doubling time: The time required for a complete fission cycle
• G = t/n where G is generation time; t is time interval in hours or minutes; n is
number of generations
• Each new fission cycle or generation increases the population by a factor of 2
• As long as the environment is favorable, the doubling effect continues at a
constant rate
• The length of the generation time- a measure of the growth rate of an organism
• Average generation time- 30 to 60 minutes under optimum conditions
• Can be as short as 10 to 12 minutes
• This growth pattern is termed exponential

12. The Mathematics of Population Growth
• The size of a population can be calculated by the following equation:
Nt = (Ni)2n where
• Nt is the total number of cells in the population, t denotes “at some point in
time”
• Ni represents the starting number of cells
• The exponent n denotes the generation number.
• 2n represents the number of cells in that generation.

13. The Mathematics of Population Growth

14. Types of culture
• Fermentation-carried out as
1. batch
2. continuous
3. fed-batch
• Dictated largely by type of product being produced

15. Batch culture
• Closed culture system – initially
contains limited amount of
nutrient
• No growth – lag phase – time of
adaptation
• Growth rate increases – grow
constantly at maximum rate – log
or exponential phase

16. Batch culture – Deceleration phase
• Cessation of growth – due to depletion of some essential nutrient in
medium (substrate limitation)
• Accumulation of some autotoxic product of organism in medium
(toxin limitation)
• Or combination of both

17. Batch culture – Stationary phase/Idiophase
• Point where growth rate has declined to zero.
• Population is metabolically active – producing secondary metabolites
• Maximum population phase
• During this phase, the growth rate and death rate of cells are almost
equal.
• Depletion of a growth-limiting nutrient in the medium or
accumulation of toxic byproduct or secondary metabolite

18. Death phase
• The curve continues to dips downward
• Most cellular activity stops/cells lose their viability
• Death rate overtakes the growth rate
• Endospores are formed and released from the parent cells by lysis or
other mechanisms

19. Batch culture – used for?
• Producing biomass – fastest growth rate and maximum cell
population
• Primary metabolites – extend exponential phase. Growth associated
production e.g. ethanol
• Secondary metabolites – decreased growth in log phase. Non growth
associated production. E.g. antibiotics
• Main carbon is exhausted and a secondary carbon source is used

20. Batch culture
No exchange of liquid medium.
• With addition of inoculum, culture grows uncontrolled until some
nutrient is exhausted.
• This is easy to set up.
• Problems/challenges:
• Nutrients not renewed
• Exponential growth of cells is limited

21. Batch reactor

22. Continuous Culture (chemostat)
• Addition of fresh medium to the vessel – exponential growth
• Medium is substrate limited
• Overflow device – added medium displaces equal volume of culture –
continuous production of cells
• Formation of new biomass balanced by loss of cells from vessel
• Chemostat culture – Cells and spent medium are continuously
removed
• State of culture is dependent upon flow rate of fresh medium

23. Continuous culture system

24. Continuous/Chemostat
• Advantages:
• Bacterial cultures can be maintained in a state of exponential growth over
long periods
• Relieves the insufficiency of nutrients
• Relieves the accumulation of toxic substances
• Relieves the accumulation of excess cells in the culture
• Problems – Imperfect mixing and wall growth
• Imperfect mixing – increase in degree of heterogeneity in fermenter
• Wall growth – Organism adheres to inner surfaces of reactor – increases
heterogeneity
• Limited by coating inner surfaces of vessel with Teflon

25. Perfusion/Turbidostat
• Perfusion culture
• Medium is pumped continuously
• Cells are retained
• Becoming popular for large-scale production
• Attains high cell density
• Cell separator

26. What is Fed-batch culture?
• In open system/Fed-batch culture – involves controlled nutrient
feeding
• Partial media changes at regular intervals
• Initial batch cultures – fed continuously or sequentially with medium
at the end of log or beginning of stationary phase
• No removal of culture fluid
• Three types – Variable volume, Fixed volume and Cyclic fed-batch

27. Fed-batch culture
• Batch culture is fed in following ways
• Same medium and concentration used to establish batch culture is
added – Variable volume
• Concentrated solution of limiting substrate is added at a rate less than
initial
• Very concentrated solution of limiting substrate is added at lesser rate
than initial – Fixed volume

28. Fed-batch culture
• Advantages:
• Suitable for cultures in which a high concentration of substrate is
inhibitory to cell multiplication and biomass formation.
• Used for biomass
• Primary metabolite
• Secondary metabolite

29. Large scale microbe production
• Problems in large scale production of microorganisms:
• Bacteria need to:
1. Be kept at the right temperature
2. Have nutrients supplied i.e. food, oxygen
3. Have waste products removed i.e. waste and carbon dioxide

30. Upstream Processing
• Three main areas:
• Producer microorganism. This include processes for
• obtaining a suitable microorganism
• strain improvement to increase the productivity and yield
• maintenance of strain purity
• preparation of suitable inoculum
• Fermentation media
• Fermentation Process

31. DOWNSTREAM PROCESSING
• This expression refers to the techniques, which need to be carried out
after growth.
• Separation and purification of product from the culture medium for
further processing.
• This is achieved by various techniques e.g. centrifugation, filtration or
flocculation of the cells, sedimentation, chromatography,
electrophoresis, etc.
• The processes that follows fermentation: Cell harvesting, Cell
disruption, Product purification from cell extracts or the growth
medium

32. Environmental factors affecting growth
Temperature and Microbial
Growth
• Cardinal temperatures
• minimum
• optimum
• maximum
• Temperature is a major
environmental factor controlling
microbial growth.

33. Classification of Microorganisms by
Temperature Requirements

34. pH and Microbial Growth
• pH – measure of [H+]
• Each organism has a pH range and a pH optimum
• Acidophiles – optimum in pH range 1-4 e.g. Helicobacter pylori, Thiobacillus
thiooxidans
• Alkalophiles – optimum in pH range 8.5-11 e.g. Vibrio cholera
• Most of pathogenic bacteria are neutrophiles
• Lactic acid bacteria – 4-7
• Thiobacillus thiooxidans – 2.2-2.8
• Fungi – 4-6
• Internal pH regulated by BUFFERS and near neutral adjusted with ion pumps
• Human blood and tissues has pH 7.2+0.2

35. Oxygen and Microbial Growth
• Utilization of O2 during metabolism yields toxic by-products including
O2
-, singlet oxygen (1O2) and/or H2O2.
• Toxic O2 products can be converted to harmless substances if the
organism has catalase (or peroxidase) and superoxide dismutase
(SOD)
• SOD converts O2
- into H2O2 and O2
• Catalase breaks down H2O2 into H2O and O2
• Any organism that can live in or requires O2 has SOD and catalase
(peroxidase)

36. Oxygen and Microbial Growth
• Aerobes :
• Obligate : require oxygen to grow
• Facultative : can live with or without oxygen but grow better with oxygen
• Microaerophiles : require reduced level of oxygen
• Anaerobes :
• Aerotolerant anaerobes : can tolerate oxygen but grow better without
oxygen.
• Obligate : do not require oxygen. Obligate anaerobes are killed by oxygen

37. Applications of microbial culture
• Production of whole microbial cells such as food like cheese and wine; and
vaccines.
• Production of primary metabolites such as acids, alcohol, enzymes, and
microbial polysaccharides.
• Production of secondary metabolites such as antibiotics and
biodegradable plastics.
• Microbial leaching of metals, effluent and waste treatment.
• Microbial cells such as agents of biotransformation of organic compounds.
• As host cells for the production of recombinant proteins, gene cloning, and
other molecular biology research.