Transcript Microorganism: a microscopic biochemical factory

Think whole process..
Industrial Production of
Bioproducts
• As soon as possible (initial start)
• As fast as possible (rate)
• As much as possible (concentration, yield)
• As long as possible (duration)
• As cheap as possible (cost)
• As simple as possible (ease of work)
• As desirable as possible (regulatory issues)
Improving Productivity
of Biologicals
• Strain improvement
• Media development
• Equipment Design
• Process Design & Control
you need to be well aware of
the needs of m/o’s
Depending on the purpose:
Different Levels of Downstream Processing
A bioreactor: is a reactor system
used for the culture of
microorganisms.
• They vary in size and complexity
from a 10 ml volume in a test tube
to computer controlled fermenters
with liquid volumes greater than
100 m3.
• They similarly vary in cost from a
few cents to a few million dollars.
Factors related to bioreactor
operation
• Product yield
• Annual demand for product
• Number of vessels
• Ease of operation and handling
• Vessel configuration
• Mode of operation
• Monitoring and control
• Safety
Standing cultures - Fernback flasks
3 liter "Fernback" flask containing 1 liter of medium and a 250
ml Erlenmeyer flask containing 100 ml of medium.
Shake flasks
Shake flasks are commonly used for small scale cell cultivation.
– Continuous shaking of the culture fluid.
– Shaking continually breaks the liquid surface and thus provides a greater
surface area for oxygen transfer.
• Higher oxygen transfer rates can be achieved with shake flasks than
with standing cultures
What about baffled and unbaffled ones..
Shake flasks- baffles
The presence of baffles in the flasks
will further increase the oxygen transfer
efficiency, particularly for orbital
shakers.
Unbaffled flask vs Baffled flask
Note the high level of foam formation in
the baffled flask due to the higher level
of gas entrainment.
Stirred bioreactors (STR)
For aeration of liquid volumes greater than 200
ml, various options are available.
Non-sparged mechanically agitated bioreactors
can supply sufficient aeration for microbial
fermentations with liquid volumes up to 3
litres. However, stirring speeds of up to 600
rpm may be required before the culture is
not oxygen limited.
In non-sparged reactors, oxygen is transferred
from the head-space above the fermenter
liquid. Agitation continually breaks the liquid
surface and increases the surface area for
oxygen transfer.
In sparged reactors, both mechanical agitations
and air sparged.
Stirred Tank Reactor
A typical mechanically agitated
and aerated bioreactor used
for microbial fermentations is
shown in the following figure:
Laboratory scale bioreactors with
liquid volumes of less than 10
litres are constructed out of
Pyrex glass.
For larger reactors, stainless steel
is used.
Modern mechanically agitated bioreactor will contain:
An agitator system
An oxygen delivery system
A foam control system
A temperature control system
A pH control system
Sampling ports
A cleaning and sterilization system.
A dump line for emptying of the reactor
Turbulent and laminar flow
Turbulence is characterized by the
formation of randomly moving
turbulent eddies.
Turbulence breaks the liquid flow lines
and leads to good mixing of the
fermenter fluid.
At low stirring speeds, a phenomenom
known as circulation or swirling
occurs. During circulation, the fluid
follows the laminar flow lines, moving
in circles around the tank; the result
being ineffective mixing .
In an unbaffled tank, as the stirrer speed
is increased, the liquid will continue to
circulate, although at a faster speed.
Eventually the liquid and air will be
drawn into impeller leading to a
phenomom known as vortexing.
•
Impellers and STR
Flow Patterns
Examples of bioreactor designs
• Stirred tank reactor (STR)
• Bubble column
• Air-lift reactor
• Packed bed
• Acetators and Cavitators
• Cyclone Column
• Cylindero-Conical Vessel
• Deep Jet Fermenter
• Rotating Disk Fermenters
• Paddle wheel reactor
• Basket reactor
• Spinfilter perfusion reactor
• Tray reactor
• Membrane Reactors
Bubble driven bioreactors
Sparging without mechanical
agitation
Fludized Bed reactors..
Two classes of bubble driven
bioreactors are bubble column
fermenters and airlift fermenters.
An airlift fermenter differs from
bubble column bioreactors by the
presence of a draft tube
Fixed bed reactors: the cells are immobilized by
absorption on or entrapment in solid, non-moving
solid surfaces
Adsorption (plastic blocks, ceramic blocks, wood
shavings or fibrous material, etc) : Once steady state
has been reached there will be a continuous cell loss
from the solid surfaces. These types of fermenters
are widely used in waste treatment.
Covalent immobilization or Entrapment: the cells are immobilized in
solidified gels such as agar or carrageenin: the cells are physically
trapped inside the pores of the gels and thus giving better cell
retention and a large effective surface area for cell entrapment.
Industrial applications of fixed bed reactors include:
• waste water treatment
• production of enzymes and amino acids
• steroid transformations
Fluidized bed reactors: the cells are
immobilized on or in small particles.
The use of small particles increases the
surface area for cell immobilization
and mass transfer. Because the
particles are small and light, they can
be easily made to flow with the liquid
(ie. fluidized).
The fluidization of the particles in the
reactor leads to the surface of the
particles being continuously turned
over. This also increases the mass
transfer rate.
Two parameters:
Minimum Fluidization Velocity and Terminal Velocity
Fluidization:
Solids will be suspended when the pressure drop
exceeds the weight of solids
Happens when the gas velocity exceeds
the minimum fluidization velocity: umf
As the velocity increases above fluidization velocity:
bed expansion..
Individual particles are blown out of the bed when the gas
velocity exceeds the terminal velocity (Free Fall Velocity)
Comparing fluidized bed and fixed reactors
Fluidized bed reactors are considerably more efficient than fixed bed reactors
for the following reasons:
The higher levels of mixing in the reactor  Mass transfer rates are higher
Fluidized bed reactors do not clog as easily as fixed bed reactors.
Particle compression in fluidized bed reactors are less than fixed bed ones
Fluidized bed reactors are however more difficult to design than fixed bed
reactors.
Cell density per unit reactor volume is less in fluidized bed reactors..
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Design considerations include:
Setting the flow rate to achieve fluidization
Ensuring that bubble size remains small during the fermentation.
Prevention of the cells from falling or "sloughing" off the particles.
Minimizing particle damage.
Size and shape of particles
Flocculated cell reactors: the cells are
trapped in the reactor due to an induced or
natural flocculation process. In flocculation
cells tend to group together causing them to
come out of solution and to sink towards the
base of the reactor.
Flocculated cell reactors are used widely in
anaerobic waste treatment processes.
In these reactors, the methanogenic and other
bacteria form naturally flocs. The flocs move
due to the release methane and carbon
dioxide by the cells.
Large scale anaerobic flocculated cell systems,
known as Upflow Anaerobic Sludge Blanket
processes are widely used
Cell recycle systems
In a fermenter with cell recycle the
cells are separated from the
effluent and then recycled back
to the fermenter; thus minimizing
cell removal from the fermenter :
Cell recycle is used in activated
sludge systems.
Biomass recycling for product or
biomass production is more
difficult due to the need for
maintaining sterility during cell
separation. Centrifugation which
is a faster process than settling
would be used to separate the
cells.
Microorganism: a microscopic biochemical factory
• C, N, O and others are IN
– to the various constituents of the cell
– to biochemical products which may be retained or transported back into
the environment outside the cell
• Metabolic activities inside the cell are regulated at various levels
both inside and outside the cell
• Biological activity is extremely sensitive to the environment too
• Because of this multi - level complex regulation, by an engineering
point of view, it is of utmost importance to understand the nutritional
and environmental factors affecting cell metabolism
Parameters characterizing a bioreactor
The presence of the living microorganisms inside the bioreactor makes it
more complicated than the conventional chemical reactor
Physical parameters
• agitation power, agitation speed, broth volume, color, expanded broth
volume (density), foaming, gas flow rate, gas humidity, heat generation rate,
heat transfer rate, liquid feed rate, liquid level, mass, osmotic pressure,
pressure, shear rate, temperature, turnover time, and viscosity.
Chemical Parameters
• amino acids, carbon dioxide (gas), cation level, conductivity, inhibitor,
intermediate(s), ionic strength, nitrogen (free and total), nutrient
composition, oxygen, pH, phosphorous, precursor, product, redox and
substrate
Parameters characterizing a bioreactor
Biochemical (intracellular) parameters
• Intracellular parameters that indicate the metabolic state
of the cell at any given time during the cell growth:
amino acids, ATP/ADP/AMP, carbohydrates, cell mass
composition, enzymes, intermediates, NAD/NADH, nucleic acids,
total protein, and vitamins
Biological parameters
• Biological parameters characterize the bioreactor in terms of what is
happening inside the reactor at the cellular level:
age/age distribution, aggregation, contamination, degeneration,
doubling time, genetic instability, morphology, mutation, size/size
distribution, total cell count and viable cell count
First: Growth..
The orderly increase in all chemical
constituents of the cell which for unicellular
organisms ultimately leads to an increase
in the number of individuals in population
The form of growth depends on the
conditions and the medium:
Liquid suspension culture, solid medium,
immobilized system??
First: Growth
One of the basic tools in microbiology
Now it is the indispensable tool in all fields of microbiology, physiology,
genetics, ecology or biotechnology...
Golden age with all those key publications:
• Monod (Nobel Prize in Physiology or Medicine in 1965, Lac operon:
the first example of a transcriptional regulation system)
• Hinshelwood (chemical kinetisist solving problems related to
bacterial physiology, papers in 46 and 66, Nobel Prize winner)
• van Neil (chemical basis of photosynthesis-purple-green bacteria,
chemistry of denitrification)
• Novick and Szilard: apparatus for the continuous culture of bacteria,
which they called a chemostat (1950)-mutation rate analysis,
negative feedback regulation.
• Pirt (antibiotic production-microbial growth dynamics and the control
of microbial processes; concept of "maintenance energy", in other
words, energy that is not required for microbial growth, but for
preserving essential cellular functions)
Growth in cell populations..
• Closed system.. Batch (kesikli)
• Semi-closed system..Fed-batch (kesiklibeslemeli)
• Open culture.. Continuous culture
(sürekli):
– Chemostat, turbidostat (homojen karışım
sağlanır)
– Plug-flow (Tapa-akışlı reaktörler) reactors
Basic Concepts
• Batch Operation
– Cultivation starts at t=0 (aşılama) and is
stopped at time t=t’.
– First proliferation of cells under non limiting
conditions
– Later higher cell density passing into
substrate-(nutrient or oxygen) limited
operation
Extented Culture Operation
• Describes the mode of operating reactor
– Concentration of the limiting substrate kept
constant by suppyling it..
– Continuously??
Continuous Operation
• Continuous stream of nutrient solution
supplied to the reactor..
• Discharge of a harvesting stream of the
approximately the same magnitude..
• Steady state reached with theoretically
constant cell denstiy and constant
concentrations
Measurement of growth
• Direct Methods (viable or total):
– Cell number density: Hemocytometer, Plates
containing agar media, Agar gel medium
placed on a microscope slide, particle
counters, Most probable number metd...
– Cell mass: CDW, OD, Turbidity
• Indirect Methods: S
– Substrate consumption, protein
measurement, ATP concentrations, viscosity,
products of cell metabolism
Study of growth
• Identify carbon and energy source
• Determine effect of antimicrobials
(antibiotics, synthetic drugs, etc.).
• Determine effect of environmental factors
(pH, moisture, temperature, mixture
cultures, etc.)
Growth
1.) yield (verim)- increase in cell number and
mass
– how many different yields?
2.) rate (hız)- change in growth per unit time
– how many different rate equations?
– Generation time or doubling time - time
required to form two cells from one cell
(exponential growth)
Importance of generation time
• Predict the concentrations of bacteria in a
food source.
• What about microbial growth and
biodegradation kinetics??
• Doubling for E. coli is every 20 mins.
after 20 generations one cell =>1 million
cells. After 10 hrs= 1 billion cells.
• Binary fission – most bacteria divide by
this process
• Other forms cell division
– Budding (most yeast, including S. cerevisiae)
– Aerial spore formation (e.g. Actinomycetes)
– Fragmentation (e.g. Fungus)
Cell Growth in Batch Cultures!!!!
• Batch culture: Growth in closed system or
environment
• Cells grow until some essential component
of medium is exhausted or the
environment changes because of
accumulation of a toxic product
Could it be same for a continuous culture?
Typical batch growth curve: Lag, Log,
Stationary, Death phases..
Lag phase
• Cells are adapting to environment.
• Intense metabolic activity (DNA and
enzyme synthesis)
• Little or no cell division
• Period of lag phase: size of inoculum, age
of inoculum, medium composition
Log phase
• Cell division (logarithmic increase or
exponential increase)
• Generation time is constant=log plot of
growth is a straight line.
• Most metabolic activity (catabolism and
anabolism)
• Cell is sensitive to adverse conditions
(antibiotics, changes in environmental factors)
Stationary phase
(Durgun faz)
• Number of dividing cells =number of death
cells.
• Lack of nutrients, accumulation of waste
products, pH changes.
• Net growth rate is zero..Cells still active
and produces the secondary metabolites..
Death phase
• Number of dying cells > number of dividing
cells
• Can be a logarithmic decrease
• Depending on the species, some cells can
survive long periods of time.
Exponential Phase Growth
• Log phase growth is first order, ie
Growth rate  to population size
• So lnX vs. t is linear, slope = m
m units are 1/t (i.e. hr-1)
dX
 X
dt
Monod Growth Kinetics
• Relates specific growth rate, μ, to substrate
concentration
• Empirical---no theoretical basis—it just “fits”!
• Have to determine mmax and Ks in the lab
• Each m is determined for a different starting S

S

Ks  S
max
Monod Growth Kinetics
• First-order region,
S << KS, the equation
can be approximated
as  = maxS/Ks
• Center region, Monod
“mixed order” kinetics
must be used
• Zero-order region,
S >> KS, the equation
can be approximated
by  = max
S << KS
mixed order
max
, 1/hr
S, mg/L
S >> KS
Determining Monod parameters
• Double reciprocal plot (Lineweaver Burk)
– Commonly used
• Other linearization (Eadie Hofstee)
– Less used, better data spread v   K v  V
m
max
[S ]
• Non-linear curve fitting
– More computationally intensive
Chemostat:
CFSTR (Continuous
Flow Stirred Tank Reactor)
for Microbial
Growth
Steady state in continuous
operation mode
• Number of m/o’s out should be balanced by the growth
volumetric flowrate (l/s)
D= dilution ratio =
 D= F/V
Volume of bioreactor
• If flowrate  WASH-OUT
• If flowrate  Substrate availability decreases
Monod vs. Michaelis-Menten:
differences
• Monod
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–
Growth
Empirical
Ks
, 1/t
• Michaelis Menten
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–
–
–
No growth; constant E
Derived from theory
Km
v, mg/L-t
Similarities are shape of curves, form of function, parameter
estimation techniques.
Product formation
i. Primary metabolites
• Product formed during the primary growth (exponential)
phase, e.g. ethanol
• Growth dependent
ii. Secondary metabolites
• Products formed near the end of the growth (or
stationary) phase, e.g. antibiotics
• Not essential for growth and reproduction
• Highly dependent on growth conditions, repression
frequently occurs
• Usually produced from primary or intermediate
metabolites, not the substrate
Media For Industrial Fermentations
The main factors:
1. Cost and availability: ideally, materials should be inexpensive, and of
consistent quality and year round availability
2. Ease of handling in solid or liquid forms, along with associated
transport and storage costs, e.g. requirements for temperature control.
3. Sterilization requirements and any potential denaturation problems.
4. Formulation, mixing, complexing and viscosity characteristics that may
influence agitation, aeration and foaming during fermentation and
downstream processing stages.
5. The concentration of target product attained, its rate of formation and
yield per gram of substrate utilized
6. The levels and range of impurities, and the potential for generating
further undesired products during the process
7. Overall health and safety implications
What is the Role of Media in Fermentation
•
In most industrial fermentation processes there are several stages where
media are required: several inoculum (starter culture) propagation steps,
pilot-scale fermentations and the main production fermentation
•
The technical objectives of inoculum propagation (The preparation of a
population of m/o’s from a dormant stock culture to an active state of growth
that is suitable for inoculation in the final production stage) and the main
fermentation are often very different  differences in their media
formulations
•
Where biomass or primary metabolites are the target product, the
objective is to provide a production medium that allows optimal growth of
the m/o
•
Secondary metabolite production is not growth related. Consequently,
media are designed to provide an initial period of cell growth, followed by
conditions optimized for secondary metabolite production. At this point the
supply of one or more nutrients (carbon, phosphorus or nitrogen source)
may be limited and rapid growth ceases
Problems Frequently Encountered
• Compounds that are rapidly metabolize may repress
product formation. To overcome this, intermittent or
continuous addition of fresh medium may-be carried out
to maintain a relatively-low concentration that is not
repressive
• Certain media nutrients or environmental conditions may
affect the physiology, biochemistry, and morphology of
the microorganism. In some yeasts the single cells may
develop into pseudo-mycelium or flocculate, and
filamentous fungi may form pellets. This is not desirable
as it affects the product yield
• The media adopted also depend on the scale of the
fermentation.
– For small-scale laboratory fermentations pure chemical are often
used in well defined media
– However, this is not possible for most industrial-scale
fermentation processes, simply due to cost, as media
components may account for up to 60-80% of process
expenditure
• Industrial-scale fermentations primarily use cost-effective
complex substrates, where many C and N sources are
almost undefinable. Most are derived from natural plant
and animal materials, often byproducts of other
industries, with varied and variable composition
• Carbon Sources
Factors influencing the choice of C-source
• The rate at which C-sources metabolized
• Price & availability
• Media sterilization
Carbohydrates: Starch from cereal, grains and maize; malt from
barley; sucrose from sugar cane; impure form: cane molasses, corn
steep liquor, whey from diary industry
Oil & Fats: Fatty acid contents (also antifoaming properties))
Hyrdocarbons and their derivatives: n-alkanes for production of
organic acids, aminoacids, vitamins
Carbon Sources-Examples
• Molasses, a byproduct of sugar production, is one of the cheapest
sources of carbohydrate. Besides a large amount of sugar,
molasses contains nitrogenous substances, vitamins, and trace
elements. However, the composition of molasses varies depending
on the raw material used for sugar production
• Malt extract, an aqueous extract of malted barley, is an excellent
substrate for many fungi, yeasts, and actinomycetes. Dry malt
extract consists of about 90-92% carbohydrates, and is composed of
hexoses (glucose, fructose), disaccrides (maltose, sucrose),
trisaccharides (maltotriose), and dextrins. Nitrogenous substances
present in malt extract include proteins, peptides, amino acid,
purines, pyrimidines, and vitamins
• Nitrogen Sources
Factors influencing choice of nitrogen sources
• Individual nitrogen sources influence the metabolic regulations (eg:
A. Nidulans, ammonia regulates the production of proteases)
• Type, concentration (NH4Cl-NH4OH, strong pH changes)
• Complex nitrogen sources favors antibiotic production
• Downstream effects
Common ones
Soybean meal (brings gradual breakdown & prevents the accumulation
of ammonia ion), ammonia, ammonium salts or nitrates
• Minerals
Require minerals for growth and metabolism
Mg, P, K, S, Ca etc. Need to be added as distinct
components
Cu, Fe, Mn, Zn, Mo etc critical for secondary
metabolite production
• Water
Mineral content & demineralization
• Precursors and Metabolic Regulators
Regulating production rather than promoting
growth
Precursors: PAA (phenylacetic acid) for Pen G; Cl for
chlortetracycline
Inhibitors: For accumulating a metabolic intermediate
eg: product: Glycerol, inhibitor: Nabisulphite, effect: acetaldehyde
production repressed, m/o: S.cerevisiae
product: tetracycline, inhibitor: bromide, effect: chlortetracyline
formation repressed, m/o: Streptomyces aureofaciens
Inducers:Majority of enzymes are inducible.
foreign protein restrict cell growth, induction at
certain concentrations
• Oxygen Requirements
Medium influence oxygen availability
Fast metabolism: Oxygen limitation
Rheology: Viscosity & its subsequent behaviour wrt aeration & agitation
Antifoams: Surface active agents reducing oxygen transfer rate
• Oxygen is usually a limiting nutrient due to its low solubility in culture
media
• While blending uniformity is essential for oxygen distribution in the
bioreactor, bubble size distribution is the most important factor for
governing mass transfer
• When bioreactors are scaled up from laboratory to production size,
their design must meet both oxygen distribution and oxygen mass
transfer requirements
Oxygen Requirements
• Important parameter in pO2 control is mixer's rotational
speed n: nmin and nmax. It means that, when controlling
pO2, n will vary only within this range. These limits are
determined in connection with eliminating of different
undesirable phenomena:
– nmin choice is determined:
• to secure the minimal partly turbulent mixing level;
• by the guaranteed bubble dispersion;
• by the prevented sedimentation.
– nmax choice is determined by:
• setting in of the intensive foaming regime;
• irreversible mechanical damages of cells;
• liquid surface fluctuation and evaporation.
Foaming
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Removal of cells from culture
Physical changes
Reduction in working volume
Lower mass & heat ratios
Invalid process data
Decrease in sterility
3 ways to solve:
Defined media, antifoams or mechanical foam breakers
Ideal antifoam
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Should disperse easily and have fast action on foam
Should be active at low concentrations
Should be long acting in preventing new foam formation
Should not be metabolized by m/o
Should be nontoxic to m/o
Should not cause any problem in the extraction step
Should be cheap
Should be sterilazable
Examples: Silicones, sulphonates, esters, fatty acids, alcohols
Media Optimization
Aim: Optimized productivity
– Different combinations and sequences of
process conditions need to be investigated
– Fixing one and varying all the others
3 nutrients at 2 concentraions: 23 trials
6 nutrients at 3 concentrations: 36 trials
Min number of experiments for optimum conditions
How can you design your media concentrations?
Isolation, Preservation and Improvement of
Industrially Important Micro-organisms
Diversity of m/o:
The power of nature for the
commercially valuable products
Screening
1st Step: Isolation of Pure or Mixed Cultures from nature
2nd Step:Assesment to determine the capacity for the
production
Criteria in the choice of organism:
1. The nutrional characteristics of the organism
2. The optimum temperature of the organism
3. The toxicity of organism and the product
4. The suitability of the organism
5. The stability of organism
6. The productivity of organism
7. The ease of product recovery from culture
Can the culture collections be an answer?
Very well known ones:
American type Culture Collection
National Collection of Type Culture
National Collection of Yeast Culture
Collection of mycological Institute
Japan Collection of Microorganisms
ATCC
NCTC
NCYC
IMI
JCM
What should we isolate and from where ?
Ecological approaches
1.
List the group of m/o to be isolated
2.
Define the ecosystem from where the samples will be isolated; mountain or
desert ?
3.
Group samples into types, for example plant, plant parts, soil, rock, water,
thermal sources…
4.
List the environmental parameters to be considered; pH, salinity, temperature..
5.
Consider constraints in terms of scale-up conditions
6.
List the available substrates in the ecosystem
7.
Design isolation techniques around data from 1-6; substrates, incubation
conditions, etc.
8.
Modify known procedures according to the material to be examined
9.
Use specific enrichment methods for microbial groups of interest
Isolation methods utilizing the selection of desired characteristics
Most important technique : Enrichment culture: The recognition that microorganisms
in nature always exist in a mixture demanded that for the study of various
physiological types, pure cultures were essential. Realising this, S.Winogradsky
(1856-1953) and M.W. Beijerinck (1851-1931) developed the technique of
"enrichment culture".
Enrichment culture : Result in increase of number of the organism relative to the
number of the others in the original inoculum
Starting from a mixed population reaching to a single organims!!
Enrichment liquid culture :
Carried out in shake flasks
Subculturing carried out for selection of dominant organism
Spreading out on a solid medium
Enrichment in solidified media : Especially useful for certain enzyme procedures
Involve the use of selective medium w/ substrate supplement to encourage growth
Choice of starting material is important, maybe preliminary tests
Ex : Alkaline protease producers ; sample spread on agar pH 9-10 clear zone +
1.
Chemical Strategies, include using a specific energy source
such as sulfate that enriches for organisms with specific abilities
to utilize it as an energy source. Controlling pH would be another
method, by keeping pH between 4.0 and 5.4 you could select for
acidophilic organisms
2.
Physical Strategies, include incubating at high or low temperature
which would select for Thermophilic (heat loving) or Psychrophilic
(cold loving) organisms. Incubating without oxygen would be
another strategy this would select for anaerobic organisms
3.
Biological Strategies, include using a live host to enrich for a
particular type of bacterial virus
Isolation methods NOT utilizing the selection of desired characteristics
If the products does not provide advantage for isolation; i.e. antibiotic producing
organisms
Isolate a pool of microorganism, test for desired characteristic
Problem : Reisolation of well-known strains
Remedy : 1- Develop procedures to favor isolation of unusual taxa
2- Identify selectable features of unselectable industrial trait for developing
an enrichment procedure
Can also :
•
Check the databases not to reisolate the strains
•
Incorporate antibiotics to select resistant taxa
Preservation of industrially important m/os
Isolation: Long + expensive procedure
it is essential to
– keep the desirable characteristics
– eliminate genetic change
– retain viability
– protect against contamination
Avoid subculturing or keep it min:
Small probability of having mutations at each cell
division, repeated sub-culturing carries the risk of
contamination..
Storage at reduced T:
4C or -20C: keeping more than 6 months may cause change in
growth pattern.....
Storage under liquid nitrogen
Metabolic activity reduced considerably by storage at low Ts :
- 150C-190C
have universal applicability fits well to all organisms
Use of cryoprotective agent and storing in sealed ampoules
Storage under deep freezing
-70C, use of cyroprotective agent..
Storage in dehydrated form
Dried Cultures .. Sterile soil for sporulating myceial organisms
silica gels, porcelain beads, filter papers
Lyophilization or freeze –drying
lyophilization followed by its drying under vacuum:
sublimation of the cell water..
Growing culture to its max. Stationary phase
Resuspending in a protective media (serum or sodium
glutamate)
common for service culture collection
Since once the culture is dry, no special
attention to storage equipment..
Long reviving period..
Best: liquid nitrogen, then the lyophilization
IMPROVEMENT OF INDUSTRIAL ORGANISMS
Natural isolates : Low productivity  increase
Increase yield by 1- optimize culture medium
2- optimize growth conditions
To what extent ? Organisms max ability controlled by the genome
So  genome must be modified  increase potential yield
Then  go for improved cultural requirements
All properties of organism depend on the sum of its gene potential…
2 categories of genes:
Structural : encode for proteins a) determining biochemical capabilities of the
organism by catalyzing particular anabolic or catabolic reactions, b)
Components of cellular structures
Regulatory : control the expression of the structural genes by determining
the rate of production of their protein products in response to intra or
extracellular signals
Sustainability
We live in a world of limited sources, with a fast-growing population
and increasing environmental problems
 Besides economical aspect, environmental and social considerations
should also be kept in mind
– Sustainability connects these 3 aspects
• More specifically, industry is sustainable when it is:
– Economically Viable - that is, it uses natural, financial, and human
capital to create value, wealth, and profits.
– Environmentally Compatible - that is, it uses cleaner, eco-efficient
products and processes to prevent pollution, as well as depletion of
natural resources, biodiversity, and wildlife habitat.
– Socially Responsible - that is, it behaves in an ethical manner and
managing the various impacts of its production
(http://strategis.ic.gc.ca/epic/site/ind-dev.nsf/en/de00005e.html)
Sustainability
• In 1713, HC von Carlowitz (German forestry): not to cut more timber
in a certain year than was added to the stock by the natural growth
• In 1987, World Commission on Environment and Development
(Brundtland Report): contemporary discussion about sustainability
– “The development that meets the needs of the present without
comprimising the ability of the future generations to meet their own
needs”
– Sustainability does not mean to preserve but to develop responsibly
• In 1999, Dow Jones Sustainability Indices were started
– Corporate sustainability is considered a business approach embracing
oppurtunities and managing risks deriving from economical,
environmental and social development (pillars of sustainability)
• "Excellent environmental performance is meaningless if no wealth is
created. Wealth in a destroyed environment is equally senseless. No
matter how wealthy, a society fundamentally lacking in social equity
cannot be sustained“ Source: Shell Report 2000
Sustainability and Bioprocesses
• Bioprocesses are becoming economically
competitive in increasing number of
industries and have advantages concerning
several local and global challenges
– Usually based on renewable sources
– Mild rxn conditions reduce risk of accidents
– By products and other wastes have normally a
low pollution potential
Impact Categories (IC) for Environmental Assessment
IC
I/O Class A
Class B
Class C
Raw Material
I
Only fossil, predicted
exhaustion in 30 yrs
Only fossil,
predicted
exhaustion in 30100 yrs
Renewable or
guaranteed long
term supply
Land Use
I
> 100 m2/kg
10 m2/kg and
< 100 m2/kg
< 10 m2/kg
Critical matt’l
used
I
Heavy metals, PCB,
In sub-st. amounts No critical cmp’d
AOX used or
involved
produced in
stoichiometric amount
Complexity of
the synthesis
I
> 10 stages
3-10 stages
< 3 stages
Thermal risk
I/O
--
--
--
Acute and
chronic toxicity
I/O
---
---
---
Impact Categories (IC) for Environmental Assessment
IC
I/O
Class A
Class B
Class C
Ecotoxicity
I/O
---
---
No water hazard
Global warming
potential
O
GWP > 20
GWP < 20
No potential
Ozone depletion
potential
O
ODP > 0.5
ODP < 0.5
No potential
Acidification
potential
O
AP > 0.5
AP < 0.5
No potential
Photochemical
ozone creation
potential
O
POCP > 30 or
NOX
30 > POCP > 2
POCP < 2
Odor
O
Odor threshold
< 300mg/m3
Odor threshold
> 300mg/m3
Eutrophication
potential
O
N-content <0.2 and
P-content <0.05
Compound w/o
N and P
Organic C pollution
potential
O
ThOD > 0.2 g O2/g
substrate
ThOD < 0.2 g
O2/g substrate
N-content >0.2 or
P-content >0.05
Interaction btw
Economic, Environmental and Social Sustainability
Example on GM crops
 GM crops reduce use of pesticides, increase the amount of food
 BUT they may cause ecological damage
• Risk is difficult to predict and acceptance of new technology affects
its success
• In US, acceptance is relatively high  GM crops are widely used
• In EU, as well as direct impact on human, effect on environmental
quality as an aspect of the quality of life is an important factor for
acceptance
– This led higher constraints for GM crops usage
– Due to social factors, the economical advantage of GM crops are highly
reduced
 All 3 aspects of sustainability should be considered early in the
process development
http://www.hdb.hr/bec2008/PDF_files/Braunegg_Bulk.pdf