Industrial Microbiology INDM 4005 Lecture 3 11/02/04

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Industrial MicrobiologyINDM 4005Lecture
311/02/04
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SECTION 2

• 2. UNIT OPERATIONS
• Overview
• ? Asepsis
• ? Media
• ? Inocula
• ? Delivery systems

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2.1. ASEPSIS

• Application of directed changes to an environment
  to prevent or retard the growth of contaminating
  organisms.
• Fundamental to GOOD INDUSTRIAL LARGE SCALE
  PRACTICE (GILSP) when applying GMP to a biotech
  process.

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• AIM
• Protect system
• Protect operator and environment
• Relate degree of asepsis to cost\ potential hazard

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ASEPSIS

• (1) OPTIONS - Asepsis related to level and type
• of microorganisms present BIOBURDEN
• Eliminate
• 1. in raw materials
• 2. at each stage of process
• 3. at end of process
• (2) METHOD
• Sterilisation,
• Disinfection,
• Pasteurisation etc.

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• (3) TECHNIQUES
• Heat e.g. autoclave
• Filtration e.g. heat-labile liquid, air
• Physical e.g. irradiation
• Chemical e.g. Caustic soda, ethanol etc.
• Design e.g. laminar air-flow (LAF units)

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2.1.1. MANAGEMENT - ASEPSIS

• Design of plant - flow through isolated
  departments
• Raw materials in --gt stage 1 --gt stage 2 --gt
  product --gt out
• ? Personnel - clothing, health, hygiene
• ? Process equipment design -
• e.g fermenter design --gt asepsis
• ? Design of ancillary equipment and services
• Inoculation
• Media preparation
• Finishing operations e.g. Pasteurisation, aseptic
  packaging etc.
• ? Design of treatment/ sterilisation cycle e.g.
  temp. and duration

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Management of asepsis --gt Quality Assurance
Function

• CASE STUDY
• Devise a protocol required for QA ? Factory
  Hygiene. Identify major microbiological tests to
  monitor level of cleanliness in a factory.

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2.1.2. REACTOR DESIGN AND ASEPSIS

• 1. Vessel material --gt steam sterilisation
• 2. Entry / exit points / ports - threaded
  covers, sterilisable
• 3. Pipework - eliminate pockets, dead spaces,
  designed slopes
• 4. Valves - pinch / diaphragm best
• 5. Impeller glands / bearings require special
  seals
• 6. Gas inlet - filter input supply, non-return
  valves
• 7. Gas outlet - filter (protect environment),
  prevent back-flow
• 8. Sterilise additives e.g. antifoam, acid/alkali
  etc.
• 9. Aseptic sampling - sequence to sterilise with
  steam, cool, collect and re-sterilise
• 10. Transfer of inoculum - sequence valves etc.
• Above principles incorporated into the design of
  sterile
• operating / processing facilities - See
  additional sheet

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Inoculation of a plant fermenter from another
plant fermenter
Fermenter
Seed Tank
C
J
G
F
Steam
A B
D
E
I
H
Trap Trap
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(a) REACTOR / EQUIPMENT DESIGN

• 1. Moving metal (e.g. rotor shaft with impeller)
  in contact with fixed metal (e.g lid of
  fermenter) problem to eliminate contamination.
  REQUIRE SEALS.
• 2. The satisfactory sealing of the stirrer shaft
  assembly is essential to maintain asepsis in long
  fermentation runs.
• 3. A simple stirrer seal is shown below

Stirrer shaft
Top bearing
Yoke
Fermenter top plate
Skirt
Bottom bearing
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TYPES OF SEALS

• Packed-gland seal
• The impeller shaft is sealed with several layers
  of packing rings of asbestos or cotton yarn
• At high speeds the packing can wear away
• Difficult to sterilise
• Bush seals
• Composed of a stationary seal and a rotating seal
• Common in laboratory scale fermenters
• Mechanical seals
• Commonly used in large fermenters
• Seal is composed of two parts, one stationary the
  other rotating, with the two components forced
  together with springs
• Magnetic seals

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Reactor Design and asepsis

• 2. Valves are required to control the flow of
  liquid e.g. transfer of inoculum from seed to
  production tank.
• A wide range of valve types exist
• TYPES OF VALVE suitable for sterile uses
• ? Pinch valve (Close valve by tightening a
  flexible sleeve with pinch bars operated by
  compressed air remotely or automatically)
• ? Diaphragm (Also uses flexible closure, failure
  due to excessive handling)
• ? Ball (Consists of stainless steel ball, with a
  machined hollow centre, can operate under high
  pressure)
• Safety valves incorporated into air / steam lines
  - ensure that
• pressure does not exceed specification

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TYPES of PUMPS

• Used to transport liquids or gases
• 1. Centrifugal - fan type, impeller imparts
  velocity to stream of fluid. Often lacks force
• 2. Rotary - positive displacement with circular
  motion. Scoops the fluid from pump chamber.
  Often used as source of power e.g. hydraulic
  systems
• 3. Reciprocating - to and fro motion
• PROBLEMS
• ? Rotary /reciprocating - compression ? heat
  (problems with temp. control due to aeration in
  fermenter)
• ? Contamination (e.g. Oil) may present
  difficulty. Leakage
• ? Property of liquid
• Caustic soda corrosive, rubber-lined
  centrifugal pump
• Viscosity - e.g. keep molasses warm

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TYPES of PUMPS

• ASEPTIC PUMPING OF LIQUIDS
• ? Difficult to sterilise mechanical pump.
• Leakage problems overcome by Diaphragm pump (fig
  8.10)
• PERISTALTIC PUMP - compress pliable tubing
  between rollers, forces liquid through the tube
• ? Pump mechanism and liquid are never in contact

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• CASE STUDY
• Draw different types of seals, valves and pumps,
  comment on design of pipework and asepsis

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(b) STERILIZATION OF AIR USING FILTERS

• (1) TYPES
• PORE SIZE SMALLER THAN PARTICLES - absolute
• PORE SIZE GREATER THAN PARTICLES - fibrous
• Mechanisms of fibrous filters include
• Inertial impaction
• Interception
• Diffusion
• Electrostatic attraction
• Can be influenced by air velocity. Problem if
  wet (condensation)

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• (2) EFFICIENCY
• Given by ratio of the number of particles removed
  (N) to that present originally (N0 )
• Plot of ln N / N0 against filter length (x) will
  yield a line of slope K
• K is influenced by filter material and by linear
  velocity of air passing through the filter
• This gives the basis of calculating the required
  length and diameter of filter to give a specified
  level of protection (or contamination) .......
  see chapter 5.

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Asepsis

• All fermentation equipment must be kept
  scrupulously clean and sanitized to avoid
  contamination by microorganisms.
• Good cleaning and sanitation has become
  especially necessary with the growing use of
  non-pasteurized products and the reduced use of
  additives.
• Cleaning and sanitizing may be separate or
  combined processes.

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Cleaning Detergents

• Detergent must have the capacity to break the
  soil into fine particles and to hold them in
  suspension so that they do not redeposit on the
  cleaned surface.
• Detergents also must have good sequestering
  power to keep calcium and magnesium salts (causes
  beerstone in brewing process) in solution.
• There are two types of cleaning detergents
  alkaline or acid that are often formulated with
  surfactants, chelating agents, and emulsifiers to
  enhance the effectiveness of the detergents.
• The most effective detergents in the use today
  are formulated with alkaline solutions that have
  chelators and surfactants.

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Sanitizing Agents

• The objective in sanitizing is to reduce the
  number of microorganisms present on a surface to
  acceptable levels.
• This is done with steam treatment or chemical
  sanitizers- alkaline and acid disinfectants.

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Cleaning And Sanitation Manual

• First step in any effective cleaning and
  sanitation program is the development of a
  detailed, up-to-date manual.
• This manual should establish a systematic
  procedure for cleaning each major piece of
  fermenter equipment, listing the frequency,
  method, and materials to be used for cleaning.
• For each cleaning procedure discussed in the
  manual, the weight or volume of material used
  should be given relative to the amount of water
  used, along with the concentrations of each
  material involved.
• The manual also should include the schedule of
  routine microbiological assessments or surveys to
  evaluate the sanitary conditions and, hence, the
  effectiveness of the cleaning and sanitizing
  operation.

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Material And Corrosion Resistance

• Stainless Steel
• The corrosion inhibitor in stainless steel is
  the passive oxide layer that protects the
  surface.
• Beerstone (calcium oxalate) can cause corrosion
  if not removed, because the metal beneath the
  deposit becomes oxygen-depleted.
• Many types of stainless steel exist. The type of
  stainless steel used in fermentation equipment is
  the nonmagnetic 300 series.

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Material And Corrosion Resistance

• Copper
• Copper generally is more acid-resistant than
  alkaline-resistant.
• Copper is usually resistant to non-oxidizing
  acids such as acetic, hydrochloric, and
  phosphoric, but is not resistant to oxidizing
  acids such as nitric and sulfuric nor to
  non-oxidizing acid solutions that have oxygen
  dissolved in them.
• Alkaline detergents will blacken copper due to
  the formation of oxides.
• Commercial detergents usually contain buffering
  agents and inhibitors that prevent corrosion of
  copper.

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Material And Corrosion Resistance

• Aluminum
• Caustic cleaners react with aluminum, actually
  dissolving the metal and pitting the surface.
• The reaction with aluminum can produce a
  potentially dangerous situation, in that
  flammable hydrogen gas is produced.
• Proper ventilation is necessary under these
  conditions.
• The unsightly pitting that can occur can be a
  good harboring point for bacteria.

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Methods Of Application
Manually Small production outlets do not have the
luxury of cleaning-in-place systems and have to
manually clean and sanitize their equipment. The
choice and concentration of detergents is limited
in manual applications, given the risks to the
user. In addition, the temperature of the water
is usually limited between 48 and
50ºC. Clean-in-Place Systems Clean-in-place (CIP)
systems were developed by the dairy industry. The
simpler CIP units are usually made of at least
one stainless steel tank, a centrifugal supply
pump, stainless steel valves, a steam injection
line, and tank spraying devices.
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What Is CIP (Cleaning In Place)

• CIP widely used in all types of process
  industries.
• CIP main purpose is to remove solids and
  bacteria from vessels and pipework in the food
  and drinks processing industries.
• CIP allows process plant and pipework to be
  cleaned between process runs without dismantling
  equipment.
• It can be carried out with automated or manual
  systems and is a reliable and repeatable process
  that meets the stringent hygiene regulations
  especially prevalent in the food, drink and
  pharmaceutical industries.

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Benefits Of Cleaning In Place
Safety Production down time between product
runs is minimised Cleaning costs can be reduced
substantially by recycling cleaning solutions
Water consumption is reduced as cleaning cycles
are designed to use the optimum quantity of
water The cleaning system can be fully
automated therefore reducing labour
requirements Automated CIP systems can give
guaranteed and repeatable quality assurance
Automated CIP systems can provide full data
logging for quality assurance requirements
Hazardous cleaning materials do not need to be
handled by operators Use of cleaning materials
is more effectively controlled using a CIP system
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CLEANING IN PLACE SYSTEMS

• A CIP system can be an automated, sequenced
  system with the option of manual intervention or
  a semi-automatic system.
• All process equipment are provided with in-built
  cleaning systems to facilitate thorough cleaning.
  

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Process Control Automation

• Fermentation is a critical bioprocess and hence
  it is essential to monitor each and every step.
  The control of Cleaning In Place systems can vary
  from simple manual operation to fully integrated
  Programmable Logic Controller (PLC) controls with
  touch screen operator interfaces.
• Level 1 To control critical process parameters
  and maintain consistency in product quality.
• Level 2 PLC based Control System with Data
  Acquisition facility to control critical process
  parameters and log data.
• Level 3 PLC based control system with auto
  sequencing and data acquisition system. This
  system facilitates control of critical process
  parameters, fault Logging and with a provision of
  generating reports with an option of internet
  based remote access.

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Different Types Of CIP Systems
Single Pass systems In a single pass system new
cleaning solution is introduced to the plant to
be cleaned and then disposed to drain. In most
cases a single pass system would start with a
pre-rinse to remove as much soiling as possible.
The detergent clean and a final rinse would
follow this.
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• Recirculation systemCleaning solution is made up
  in an external tank, introduced to the plant to
  be cleaned, recirculated and topped up as
  required until the cleaning cycle is complete. In
  general recirculation systems use less water
  cleaning detergents but require greater capital
  outlay.

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System Design

• The first design consideration for a Cleaning In
  Place system is the cleaning requirement for each
  process vessel.
• Factors to be considered can include
• the size of the process vessels
• standard of cleaning required,
• the available cleaning time,
• the type of cleaning medium,
• and whether recycled detergent can be
  used.With this information cleaning heads can
  be selected to meet the requirements described
  above. This then allows pumps to be selected to
  match the flow rates required for the heads and
  the type of cleaning material being used.
• The module size and configuration can also be
  calculated from this information.

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System Design

• The process diagram on next slide shows an
  example of a three-tank Cleaning In Place system.
  
• There are three holding tanks, which are mounted
  in a stainless steel bund.
• The tanks are normally a bulk caustic tank,
  dilute detergent tank and fresh water tank.
• Bulk cleaning liquid is normally delivered to a
  connection point outside the process plant and
  pumped to the storage tank by the delivery
  vehicle.
• Heating can be specified for the bulk cleaning
  liquid tank depending on the properties of the
  cleaning agent.
• Lagging and cladding can also be fitted for
  improved energy efficiency.

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Process Control in CIP
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• The bulk cleaning liquid is pumped to the
  detergent tank before each cleaning cycle along
  with water to make up a batch of detergent.
• The detergent is normally more effective at a
  higher temperature. If this is the case it would
  then be pumped through a plate heat exchanger to
  bring it to the required temperature.
• The system shown below allows the detergent to
  be re-circulated through the heat exchanger until
  it reaches the optimum temperature.

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• CASE STUDY
• DESIGN A CIP SYSTEM FOR BREWING.
• Compare various chemical agents.
• See textbooks on Brewing

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Summary

• Bioreactor design and asepsis
• Design of sterile operating and processing
  facilities
• Importance of CIP
• Levels of CIP systems
• Process control