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BIOPROCESS ENGINEERING HOW CELLS GROW

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Title: BIOPROCESS ENGINEERING HOW CELLS GROW


1
BIOPROCESS ENGINEERINGHOW CELLS GROW?!
  • Prepared by
  • Wan Salwanis Wan Md Zain
  • A1-02-01 ext 2382

2
INTRODUCTION
  • Growth of Microbes
  • Replication
  • Change in cell size
  • Convert nutrient from medium into biological
    compounds
  • substrates cells ? extracellular product
    more cells
  • rate of growth 8 cell concentration
  • µnet 1/X dX/dT

3
BATCH GROWTH
  • Culturing a cell w/o further addition or removal
    of nutrient
  • Cell must be quantified either
  • Directly (not feasible due to presence of
    suspended solids), or
  • Indirectly (cell mass, cell number)

4
Determining Cell Number Density
  • Cell Number Density
  • Using Petroff-Hausser slide/hemocytometer
  • 20 grid squares in counted using microscope ?
    average
  • Disadvantages
  • i) Medium must be free from particles
  • ii) Stain is used to differentiate between
    dead/live
  • cells
  • iii) Not suitable for aggregated cultures
  • iv) Not suitable for molds

5
Determining Cell Number Density
  • Plate Count
  • Used for counting viable cell
  • Unit Colony Forming Unit (CFU)
  • Cultures are diluted and spread on agar surfaces
  • Plates are incubated and viable colonies are
    counted
  • A good plate count must consist between 30-200
    colonies
  • Suitable for yeast and bacteria
  • Less suitable for molds
  • Selection for best medium growth is crucial
  • Single cell ? observable colony (25 generations)

6
Determining Cell Number Density
  • Electrical Resistance of cells
  • Cells pass the orifice causes resistance and
    provide pulses
  • Number pulses is a measure ? number of cells
  • Height of pulses ? measure cell size
  • Light Intensity Measurement
  • Intensity of light  ? cell concentration
  • Only for dilute suspension

7
Determining Cell Mass Concentration
  • Direct Method
  • Biomass Determination
  • Specthrophotometer
  • Indirect Method
  • Measurement of cellular component
  • E.g ATP, enzyme, chlorophyll

8
Definitions
  • Fermentation
  • Traditionally, defined as the process for the
    production of alcohol or lactic acid from
    glucose.
  • Broadly, defined as an enzymatically controlled
    transformation of organic compound (Websters
    New College Dictionary)

9
Typical Bioprocess
Stock culture
Raw materials
Medium preparation
Microorganism cell preparation
Shake flask
Medium formulation
Seed fermenter
Sterilization
Computer control
Production fermenter
Air
Recovery
Microbiology, biochemistry
Chemical, engineering
Products
Purification
Effluent treatment
10
Growth Patterns Kinetics in Batch
  • Lag Phase
  • Exponential Phase
  • Deceleration Growth Phase
  • Stationary Phase
  • Death Phase

11
Lag Phase
  • Occur immediately after inoculation
  • Period of adaptation of cells
  • Cell mass increase, number of cell remained
    constant
  • Poor condition of inoculum (age of inoculum)
  • Low concentration of some essential nutrient
  • Inoculum size (5 10v/v)
  • Multiple lag phase diauxic growth
  • Addition of growth activator gt Mg2 Aerobacter
    aerogenes grow in glucose and phosphate

12
Exponential Phase
  • Logarithmic phase
  • Cells adjusted to new environment
  • Cells mass numbers multiply rapidly
  • Balance growth gt all component growth _at_ same
    rate
  • Growth rate independent of nutrient concentration

13
Calculation
  • First order growth rate
  • The doubling time, td

14
Calculation
  • Biomass Yield, YX/S
  • Bacteria Yx/s gt 0.4 0.6 g/g
  • Yx/o gt 0.9 to 1.4 g/g
  • Product Yield, YP/S

15
Assignment
  • A strain of mold was grown in a batch culture on
    glucose and the following data were obtained.
    Calculate the
  • A) Maximum net specific growth rate
  • B) Calculate the growth yield
  • C) Maximum cell concentration if 150g of glucose
    is used with the same size inoculum?

16
Deceleration Growth
  • Growth decelerates due to
  • Depletion of essential nutrient
  • Accumulation of toxic by product
  • Unbalanced growth gtrestructuring of cell
  • What are the essential nutrient??!!

17
Stationary Growth
  • Zero growth rate (no cell division) or,
  • Growth rate equal to the death rate
  • Cells metabolically active
  • Production of secondary metabolites occurs (eg
    antibiotics, hormones)
  • Endogenous metabolism take place

18
Stationary Growth
  • Occur due to
  • Exhaustion of essential nutrients
  • Accumulation of toxic by product
  • Few phenomena may occurs
  • Total cell mass constant, viable cell decrease
  • Cell lysis, cryptic growth occur
  • Cells not growing, active metabolism (produce
    secondary metabolism)

19
Product Formation
  • Product formation
  • Primary Metabolites
  • Growth associated
  • Secondary Metabolites
  • Stationary growth associated

20
QUIZ 1 BKC 4622
  • 1) What are the possible reasons that cause the
    microbes to enter the stationary phase.
  • 2) Describe briefly the correlation of growth of
    microbes with
  • - Production of primary metabolites
  • - Production of secondary metabolites
  • 3) List two purposes of secondary metabolites to
    the microbes.

21
Overview of Microbial Products
  • Foods
  • Breads, cheeses, yogurt, mushrooms, wine, beer,
    soy sauce, sake, etc.
  • Commodities
  • Food additives amino acids, thickening agents,
    vitamins
  • Solvents butanol, ethanol
  • Biofuels ethanol, methane, hydrogen
  • Fine chemicals
  • Pharmaceuticals antibiotics, antifungals
  • Laboratory and diagnostic reagents enzymes
    biochemicals, proteins

22
Microbial Product
  • Leudeking-Piret Equation to model the synthesis
    rate
  • Expressed in specific rates (with respect to X)
  • Classified to 3 categories-
  • A) Growth Associated
  • B) Non Growth Associated
  • C) Mixed Growth Associated

23
Growth Associated Product
  • Produced simultaneously with growth
  • Specific rate product formation 8 specific growth
    rate
  • Product Primary metabolites
  • E.g Enzyme protease
    (Bacillus subtilis), amino acid.

qp aX 1/X dP/dt YP/X mg
24
Non Growth Associated Product
  • Production occur during stationary phase
  • The specific rate of product formation is
    constant
  • Product Secondary metabolite
  • E.g Hormones, antibiotics (penicillin)

qp b constant
25
Mixed Growth Associated
  • Production occur during slow growth and
    stationary phase
  • Eq given
  • E.g xanthan gum, lactic acid certain secondary
    metabolites

qp amg b constant
26
Primary Metabolites
  • Formed during primary growth phase
  • Product essential for the metabolic activity
    growth
  • Produced from growth substrate by the cell
    activity
  • E.g Alcohol (Saccharomyces cerevisiae), amino
    acid

27
Secondary Metabolites
  • Formed at the end or during stationary growth
  • Each formed by very few orgs
  • Not essential for growth
  • Growth conditions crucial to determine the
    synthesis rate of secondary metabolites
  • Group of closely related structures
  • Can be overproduced
  • From growth substrate or primary metabolites
  • E.g Penicillin, b-lactam antibiotics
    (Streptomyces spp., Nocardia spp.,Cephalosporium
    spp.)

28
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29
Bioethanol plant in Kyowa Hakko,Japan
30
ASSIGNMENT 2
  • Browse the internet or/go to the library and look
    up some journal articles of your choice.
    Describes two different fermentation processes
    (current or potential) from materials you read.
    The following information should be included.
  •  
  • 1. Name of the journal, volume number, and page
    number.
  • 2. Name of the microorganism(s) including the
    codes if applicable.
  • 3. Products and their potential applications.
  • 4. Growth/operational conditions of the
    fermentation including media composition,
  • pH, temperature, etc.
  • Suggested journals.
  • Bioresource Technology
  • Biotechnology and Bioengineering
  • Applied Biochemistry and Biotechnology
  • Biotechnology Progress
  • Enzyme and Microbial Technology
  • Journal of Bacteriology
  • Pages 5-10, 1.5 spacing, font size 12. Submit
    your assignment before 8th June 2006.

31
Environmental Effect on Growth
  • Temperature
  • pH
  • Oxygen Availability
  • Osmotic Pressure/Salt Concentration
  • Nutrient Availability

32
Effect of Temperature
  • Effects of temperature on growth
  • Higher temperatures speed up chemical reactions,
    double rate for every 10 deg. C in temperature.
  • Expect cells to grow more rapidly as temp. rises,
    up to a point. But too high temperatures
    denaturation of proteins and nucleic acids, loss
    of critical enzymes and loss of metabolism.
  • Cardinal temperatures every organism can be
    characterized by 3 temperatures
  • minimum temperature, below which no growth occurs
  • optimum temperature, at which fastest growth
    occurs
  • maximum temperature, above which no growth occurs
  • Different microbes adapted to different
    temperature ranges
  • Typical bacterium can grow over 30 deg. C temp.
    range (stenothermal) some can grow over wider
    range (eurythermal).

Optimum Temperature for growth and product
formation can be different
33
Effect of Temperature
  • Psychrophiles -- optimum temp. typically 15 deg C
    or lower. Note some organisms are
    psychrotolerant -- optimum temperature is 20-40
    deg, but can grow as low as 0 deg. These are not
    considered psychrophiles.
  • Mesophiles -- optima from 20-45 deg, minimum
    around 15-20 deg.
  • Thermophiles -- optima 55 deg or higher. Some
    (hyperthermophiles) have optima of 80 deg or
    higher (mostly Archaea in this group). Found in
    hot springs, deep-sea hydrothermal vents, other
    locations.

34
Effect of Temperature
  • Physiological and structural adaptations are
    related to temperature
  • Psychrophiles produce enzymes with lower
    temperature optima. They often denature at room
    temperatures.
  • Psychrophiles have higher unsaturated fatty acids
    in membrane lipids, keeps membranes fluid at
    lower temperatures.
  • Thermophiles have enzymes that are heat stable,
    also ribosomes work at higher temps. Only a few
    amino acid changes from mesophile proteins seem
    necessary in some cases to allow high temperature
    stability.
  • Thermophile membranes have many long-chain fatty
    acids, lots of saturated fatty acids. membrane
    lipids "freeze" into solid form at what we would
    consider warm temperatures, thus inhibiting
    transport. But at very high temperatures,
    membranes function well

35
Effect of Oxygen
  • Oxygen- growth limiting factor
  • Above critical oxygen concentration, growth rate
    independent of dissolved oxygen (DO)
  • E.g Azotobacter vinelandii only 50 of growth _at_
    DO 0.05 mg/l (excess glucose)
  • Oxygen requirement for organisms
  • Bacteria/yeast gt 5 10
  • Molds gt 10 -50 (pellet size)
  • Oxygen Uptake Rate (OUR)
  • Oxygen Transfer Rate (OTR) gt effect reactor
    design
  • Effect of mineral salt/organic compounds on DO
    saturation???

36
Effect of Oxygen
  • Effects of oxygen on growth
  • Note higher organisms all require oxygen But
    many microbes grow anaerobically some or all of
    the time.
  • Obligate aerobes -- grow only when oxygen is
    present
  • Facultative anaerobes -- grow with or without
    oxygen, grow better in oxygen (respire)
  • Aerotolerant anaerobes -- ignore oxygen, grow
    equally well with or without
  • Obligate anaerobes -- die in presence of oxygen
  • Microaerophiles -- won't grow at normal
    atmospheric oxygen (20), but require some oxygen
    for growth (2-10)
  • Anaerobic habitats more common than expected. Ex
    in human mouth, plaque contains bacterial zoo.
    Facultative anaerobes consume oxygen, create
    anaerobic microenvironment fit for obligate
    anaerobes. In general, wherever organic matter
    accumulates, microbes will use up oxygen faster
    than it can be replaced, create anaerobic
    environment. Esp. true under water, since oxygen
    is poorly soluble in water. Lakes and ponds
    stratify into aerobic (upper) and anaerobic
    (lower) zones in summer due to vigorous microbial
    growth on sediments.

37
Effect of Oxygen
  • Why obligate anaerobes/Why excessive oxygen
    supply is lethal to certain organism?
  • Oxygen itself is reactive (oxidizing agent),
    capable of degrading organic molecules. But
    oxygen can easily generate very toxic byproducts,
    strong oxidizing agents that react
    indiscriminately with any organic molecules,
    including DNA, proteins, etc. (superoxide,
    peroxide hydroxyl radical)
  • Aerobes (and all cells able to tolerate oxygen)
    must have enzymes to get rid of these radicals.
    Superoxide dismutase and catalase are two crucial
    enzymes. superoxide (O2-) H ---(superoxide
    dismutase) O2 H2O2peroxide (H2O2)
    ---(catalase) O2 H2O Note If E. coli (a
    facultative anaerobe) is mutated so it loses
    these two enzymes, resulting mutant behaves like
    an obligate anaerobe -- good confirmation of
    idea.
  • Culture techniques
  • for aerobes shake or rotate culture to add more
    oxygen, or bubble filtered air through culture
  • for anaerobes use media with reducing agent
    (combines with oxygen chemically)pump out air,
    flush with pure nitrogen gas GasPak jar, seal
    plates in jar, use catalyst hydrogen gas to
    remove oxygen

38
Effect of pH
  • Effects of pH on growth
  • Hydrogen ion concentration effect the activity of
    enzyme, therefore affected the microbial growth
    rate
  • pH measures acidity. pH log 10 of H
    concentration.
  • Pure water has pH of 7 1 molar acid pH 0.
  • Diff microbes have diff pH optima
  • Acidophiles acid pH optimal (1 to 5.5)
  • Neutrophiles pH 5.5 to 8 optimal
  • Alkaliphiles pH 8.5 to 11.5
  • Extreme alkoliphiles optimum pH 10 or greater
  • Note most bacteria are neutrophiles (Exceptions
    some bacteria in hot springs have optimum of 1-3)
  • But most fungi prefer slight acid (pH 4 to 6)
  • Saboraud's Medium -- uses low pH to stop
    bacterial growth, selective for fungal growth.

The Optimal pH for growth and product formation
can be different
39
Effect of pH
  • Organism maintain their intracellular pH at
    constant level
  • Different organism requires different pH range
  • Supply of CO2 to medium can alter the pH of
    medium (e.g animal cell culture/seawater)
  • In fermentation, selection of medium component
    affect the pattern of pH profile during operation
  • E.g N source Ammonium (consumption, reduced
    pH)
  • Nitrate (Nitrate reduced to Ammonium,
    increased pH)
  • Optimal pH for growth for various organisms
  • Bacteria 3 8
  • Yeast 3 6
  • Molds 3 7
  • Plant Cells 5 6
  • Animal Cells 6.5 - 7.5
  • Control pH Buffer

40
Effect of Osmotic Pressure
  • Availability of Water (Osmotic Effects)This is
    not only referring to availability of water (dry
    versus wet) but also the concentration of ions or
    solutes in the available water which will effect
    its utilization. What is water activity?
  • Some organisms can deal with high salt or solute
    concentrations, and require it to survive.
  • Organisms classified into 3 categories
  • Halophiles - 1 - 15 NaCl
  • Extreme Halophiles - 15 -30 NaCl
  • Halotolerant - organisms can survive in higher
    salt concentrations but
  • do not prefer it.
  • What are the osmotic pressure of seawater?? 3
    NaCl

41
Effect of Solutes
  • Effects of solutes and water activity on growth
  • Cells require certain amount of free water to be
    able to carry out metabolism. When placed in
    hypertonic environments, many cells stop growing.
  • Some can compensate, synthesize compatible
    solutes ( molecules whose function is to balance
    osmotic strength). Examples choline, proline,
    betaine, glutamic acid, etc.
  • Staphylococci are good examples grow on skin,
    where salts are common. Staph can tolerate up to
    10 salt can design culture media with 7.5
    salt, suppress growth of most other bacteria,
    select for Staph
  • Some bacteria require very high osmotic strengths
    for growth Halophiles Ex. Halobacterium
    halobium grows in Dead Sea, Great Salt Lake,
    evaporating salt flats. Won't grow if salt
    concentration much less than 3M!
  • Note these are members of Archaea have very
    modified cell walls and membranes. Accumulate
    enormous amounts of potassium as compatible
    solute.

42
Effect of Radiation
  • Effects of radiation on growth
  • Light and UV are parts of EM spectrum extends to
    very strong radiation (gamma rays), very weak
    radiation (heat, radio)
  • Visible light (esp. more energetic violet and
    blue) are quite strong, can kill bacteria. Many
    bacteria that are spread by air are pigmented
    pigments adsorb radiation, prevent damage to
    cell.
  • Note pigment-less mutant shows much more
    sensitivity to light than pigmented form.
  • Mechanisms of damage
  • light adsorbed by some pigment (e.g. cytochrome,
    flavin, chlorophyll), energy transferred to
    oxygen to generate singlet oxygen very strong
    oxidizing agent, causes lots of damage
  • UV light causes specific damage to DNA, max.
    effect at 260 nm --gt thymine dimers
  • Ionizing radiation causes many types of damage
    breaks H-bonds, oxidizes many groups, can break
    DNA strands (most vulnerable target).

43
Effect of Nutrients
  • Nutrient factors Affecting Bacterial Growth
  • Generally the concentration of solutes (i.e.
    chemical growth components) is higher within the
    microbial cell than in the extracellular
    environment. The major barrier governing this
    differential passage of chemical components is
    the cell membrane.
  • Membrane function is i) keep essential
    nutrients and macromolecules inside the cell.
    ii) pump certain nutrients inside the cell
    against a concentration gradient. iii) permit
    free flow of nutrients across the membrane. iv)
    exclude some solutes within the environment from
    entry into the cell

44
Continuous Culture
  • The importance of continuous culture
  • Maintenance of a culture in constant
    environmental conditions through continuous
    supply of nutrient
  • Provision of nutrients and removal of wastes.
    Useful for
  • Study in a certain growth phase
  • Study under low nutrient concentrations
  • Evolution studies

45
How Cells Grow in Continuous Culture
  • Fresh medium continually supplied to well-stirred
    culture
  • Product (cell/culture medium) continuously
    withdrawn
  • During cultivation, growth product formation
    can be prolonged.
  • At steady state cell, product and substrate
    concentration remain constant.
  • An essential nutrient is in limiting quantities

46
Devices
  • Plug flow reactor (PFR)
  • Continuous cultivation
  • No backmixing
  • Chemostat
  • Refers to constant chemical environment
  • Perfectly mixed continuous flow
  • Equipped with pH, DO, level controller
  • Feeding of fresh medium and removal of cell
    suspension occur at the same rate
  • Volume of reactor constant
  • Turbidostat
  • Cell concentration in the culture vessel constant
    (monitor the OD feed flow rate)
  • Volume is kept constant by removal of culture
    broth
  • Suitable for microorganisms able to withstand
    environmental stress
  • Flow rate into the system is adjusted to maintain
    preset turbidity (cell density).
  • No limiting nutrient

47
Chemostat
  • Apparatus that feeds sterile media into a culture
    at the same rate in which it is removed
  • Essential nutrient is limiting so that flow rate
    determines growth rate
  • Dilution rate rate at which medium flows
    through vessel relative to vessel size
  • Note cell density maintained at wide range of
    dilution
  • Rates and chemostat operates best at low dilution
    rates
  • No matter how fast the media goes in the bacteria
    cannot grow faster than they would in batch
    culture under the conditions employed within the
    chamber.

48
THE MONOD EQUATION
  • Biomass growth is dependent on nutrient
    availability.
  • As a concentration of nutrient becomes growth
    limiting substrate, the specific growth rate
    reduces until growth ceases to the unavailability
    of that nutrient. A typical plot of specific
    growth rate against the concentration of a growth
    limiting nutrient is shown below
  • In 1942, Jacques Monod proposed that the
    following mathematical relationship could be used
    to describe the effect of a growth limiting
    nutrient on specific growth rate
  • where
  • µm is the maximum specific growth rate
  • Ks is the saturation or Monod constant and
  • S is the concentration of the growth limiting
    substrate.
  • Equation 13 can therefore be re-written as

49
Cell cultures can be grown on shakers or in
fermentors
50
ASSIGNMENT
51
Turbidostat
  • Regulates the flow rate of media through vessel
    to maintain a predetermined turbidity or cell
    density
  • Dilution rate varies
  • No limiting nutrient
  • Operates best at high dilution rates

52
How to make your own yoghurt
  • Boil 1-1.5 L of milk (more fat will make a
    richer end product) on the stove
  • Cool to room temperature
  • Stir in 2 tablespoons of starter culture. Mix
    well with a whisk. You can use plain yoghurt from
    the supermarket for the first batch. Make sure
    it has Acidophilus and Bifidum
  • Put in oven at F for 3 hours
  • Transfer to refrigerator overnight. Yoghurt
    should be done in the morning
  • This yoghurt has no preservatives or sugar
    therefore keep it in the fridge, eat quickly with
    jam, syrup, fresh fruit, berries etc.
  • Tastes amazing with Indian food or make tsaziki
    sauce.
  • Save a few tablespoons to use as starter culture
    for the next batch. Dont eat it if its pink,
    green or smells funny!

53
  • Importance of Continuous culture methods
  • constant supply of cells in exponential phase
    growing at a known rate
  • study of microbial growth at very low nutrient
    concentrations, close to those present in natural
    environment
  • study of interactions of microbes under
    conditions resembling those in aquatic
    environments, food and industrial microbiology

54
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55
THANK YOU
Salwanis ext 2382
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