BIOPROCESS ENGINEERING HOW CELLS GROW

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BIOPROCESS ENGINEERINGHOW CELLS GROW?!

• Prepared by
• Wan Salwanis Wan Md Zain
• A1-02-01 ext 2382

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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

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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)

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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

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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)

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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

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Determining Cell Mass Concentration

• Direct Method
• Biomass Determination
• Specthrophotometer
• Indirect Method
• Measurement of cellular component
• E.g ATP, enzyme, chlorophyll

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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)

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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
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Growth Patterns Kinetics in Batch

• Lag Phase
• Exponential Phase
• Deceleration Growth Phase
• Stationary Phase
• Death Phase

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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

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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

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Calculation

• First order growth rate
• The doubling time, td

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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

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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?

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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??!!

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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

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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)

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Product Formation

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

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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.

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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

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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

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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
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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
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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
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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

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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.)

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Bioethanol plant in Kyowa Hakko,Japan
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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.
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• 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.

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Environmental Effect on Growth

• Temperature
• pH
• Oxygen Availability
• Osmotic Pressure/Salt Concentration
• Nutrient Availability

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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
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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.

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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

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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???

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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.

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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

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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
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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

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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

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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.

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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).

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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

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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

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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

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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

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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.

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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

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Cell cultures can be grown on shakers or in
fermentors
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ASSIGNMENT
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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

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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!

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• 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

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THANK YOU
Salwanis ext 2382