MODULE 2 Flashcards
(83 cards)
1
Q
*microscopic organisms that exist as unicellular,
multicellular, or cell clusters
*widespread in nature and are beneficial to life
(but some…)
*organisms that can exist as single cells, contain
a nucleic acid genome for at least some part of
their life cycle
A
Microorganisms
2
Q
BioMeg microbial inoculant
A
Bio stimulant for improved yield and nutritional quality of sweet potato and purple yam
2
Q
What are microorganisms?
…..in terms of size
A
“giant” bacteria
2
Q
- is an endotoxin produced by gram (-) bacteria
A
LPS
3
Q
is antigenic
A
Lipid-A
4
Q
- Gram positive
- Non-sporforming
- Anaerobes, lack catalase
- Acid- and halotolerant
- Ferment glucose to only lactic acid or to lactic acid, CO2 and EtOH
- Obtain energy only from the metabolism of sugars
- Have limited biosynthetic abilities - complex media
- Streptococcus pneumoniae: meningitis, pneumonia
A
Lactic Acid Bacteria
5
Q
Nutritional Requirements of Microbial cells
A
1.Energy source
2. Carbon source
3. Nitrogen source
4. Phosphorus and sulfur source
5. Metallic elements
6. Vitamins
5
Q
Bacteria are Blank and the
remaining components have the
following elemental composition
A
90% water
6
Q
The carbon and energy sources are usually
referred to as ?
A
SUSBTRATES
7
Q
Microbial Cells:
Environmental Requirements of MCOs
A
- Temperature
- PH
- Gaseous environment
8
Q
Example of energy source
A
- Phototrophs
- Chemotrophs
9
Q
Example of carbon source
A
- Autotrophs
- Heterotrophs
10
Q
2 types of culture composition
A
- Pure culture
- Mixed culture
10
Q
Contains only one kind of microorganisms
A
Pure culture
11
Q
Contains more than one kind of microorganisms
A
Mixed culture
12
Q
Are generally done in pure cultures.
A
Industrial fermentations
12
Q
Are mixed cultures
A
Biological processes
13
Q
Recovery cost of bulk of penicillin G
A
20-30%
13
Q
Recovery cost of Industrial ethanol
A
15%
14
Q
Recovery cost of enzymes
A
70%
15
Q
Recovery cost of monoclonal ABS
A
80-90%
16
Q
The choice of recovery process is
based on the following criteria
A
- location of the product
- concentration of the product in the fermentation broth
- physical and chemical properties of the desired product
- intended use of the product
- to minimal acceptable standard of purity
- magnitude biohazard of the product or broth
- impurities in the fermenter broth
- marketable price of the product
16
Q
Factors to determine the most
appropriate under a given set of
circumstances:
A
- capital costs
*processing costs - throughput requirements
- yield potential
*product quality - technical expertise available
- conformance to regulatory
requirements
*wastewater treatment needs - continuous or batch processing
*automation
*personnel health and safety
16
Q
Techniques used to modify handling
characteristics of fermentation broth:
A
- selection of a MCO which does not produce pigments
or undesirable metabolites
*modification of the fermentation conditions to reduce the
production of undesirable metabolites
*precise time of harvesting
*pH control after harvesting - temperature control after harvesting
17
The product formed after
fermentation is the fermented
liquor that looks hazy due to
antifoaming agents, mycelia
etc.
Fermentation broth
18
Therefore, to separate these
things, a slurry of calcium
hydroxide, i.e.Ca(OH)2to form
a precipitate of calcium citrate.
The precipitate of calcium
citrate is filtered and washed.
Filtration - Precipitation (By calcium oxide)
19
After filtration, treat a filtrate
with the sulphuric acid for
calcium precipitation as
“Calcium sulphate” (CaSO4).
Dissolution (Sulphuric acid)
20
Calcium sulphate is then treated with the activated carbon, by which it gets demineralized after passing it consecutively from the ion exchange bed
(Activated carbon) - Purification - (Ion exchange columns)
21
*After the completion of these
steps, the remaining solvent
is dried, sieved and then
packed.
*The remaining mother liquor
is again recovered through
the same process.
Sieving - Citric acid product
21
* The solution obtained from
this is subjected to the
circulating crystallizers.
* The centrifugation then
removes the crystals formed
as a result of crystallization.
Crystallization
22
removal of large solid particles and microbial cells
Microbial cell recovery
22
Lab scale centrifuges, filters, chromatography equipment, etc.
LAB SCALE
22
Large equipment for production separation and purification
Large scale
23
Unit Operations for the Removal of Microbial
Cells and Other Solid Matters: (lab scale)
1.Decantation
2.Gravity Filtration
3.Suction Filtration
4.Centrifugation
24
difference in density between components
DECANTATION
24
SUCTION/VACUUM FILTRATION
Filtering precipitates after reactions, often involving fine particles
Chemical synthesis
25
SUCTION/VACUUM FILTRATION
Analyzing water samples for suspended solids
environmental analysis
26
SUCTION/VACUUM FILTRATION
Clarifying and purifying large volumes of solutions
Pharmaceutical production
27
SUCTION/VACUUM FILTRATION
Preparing solid samples for further analysis by removing excess liquids
Drying samples
28
*They can be handy for tiny labs with limited
space because of their diminutive size.
*These are compact and are frequently
employed in research and clinical
laboratories.
Benchtop or tabletop centrifuges
29
*They have a very small footprint and take
up minimal room on the workstation
because of their highly compact form.
*These work well with small tubes (up to 2.0
ml) and are frequently employed in
biological applications.
*They are used to microfilter small amounts
of aqueous samples and hold pelleted
nucleic acids, proteins from solutions, and
other substances.
Microcentrifuge
30
*These centrifuges run at their top speeds while keeping a constant temperature.
*It is used to analyze DNA, RNA, PCR, and antibodies because its temperature range is between -20 and -40 ºc
*They are frequently used to collect sedimenting materials quickly, including yeast cells, chloroplasts, and more.
refrigerated centrifuge
30
* is a type of centrifuge that can
work at somewhat faster rates ranging between 15,000 and
30,000 revolutions per minute.
* contain a device for regulating both
the temperature and speed of the operation for the critical analysis of delicate biological molecules.
*These centrifuges employ three rotors: fixed angle, swinging bucket, and vertical.
High-speed centrifuge
31
*These are frequently used in laboratories for routine particle sorting operated at a maximum speed of 4000-5000 rpm.
*There are few instances of temperature regulation, and they are often operated at room temperature.
*These centrifuges employ swinging bucket and fixed angle rotor types.
Low-speed centrifuge
31
is a chemical additive that reduces and hinders the formation of foam in industrial fermentation processes
anti-foaming agent (defoamer)
31
bubbling a gas through a liquid to remove dissolved gases or volatiles.
Sparging
32
Are most effective against gram-positive bacteria, envelope viruses, fungi
Quats
33
allows enrichment and concentration in one step thereby reducing the volume of material for further processing.
Precipitation
34
Typical agents for precipitation
1. Acids and bases
2. Salts
3. Organic solvents
4. Nonionic polymers
5. Polyelectrolytes
6. Protein binding dyes
7. Affinity Precipitants
8. Heat treatment
35
is highly soluble in cold aqueous solutions and is frequently used in “salting-out”
purification.
Ammonium sulfate
35
At low concentration, added salt usually increases the solubility of charged macromolecules because the salt screens out charge-charge interactions.
So low (salt) prevents aggregation and therefore precipitation or "crashing"
Salting In
35
At high concentrations added salt lowers the solubility of macromolecules because it competes for the solvent (H2O) needed to solvate the macromolecules.
So high salt removes the solvation sphere from the protein molecules, and they come out of solution.
Salting out
35
Another method to precipitate proteins is to use water-miscible organic solvents (change in the dielectric constant)
Organic solvents
36
a polymer, a polyether compound
derived from petroleum with many applications
Polyethylene Glycol (PEG)
37
bind to and precipitate certain classes of
protein
(triazine dyes)
38
able to bind to, and precipitate, compounds selectively.
utilizes a heterobifunctional ligand, which, in addition to having affinity for the target protein(s), possesses another function for controlling precipitation.
Affinity precipitates
38
for various thermostable products and in the deactivation of cell proteases
Heat treatment
38
is a scalable, simple and relatively economical procedure for the recovery of a product from a dilute
feedstock.
Precipitation
39
used at all scales of operation to separate suspended particles from a liquid or gas
*Using a porous medium which retains the particles but allows the liquid or gas to pass through
Filtration
40
*It represents the flow through a uniform and constant depth porous bed.
Darcy equation
41
* It is practically used when bacteria or
other fine or gelatinous suspensions
prove slow to filter or partially block a
filter.
* can improve the permeability and sometimes porosity of a filter cake, improve filtrate clarity and help to prevent filter medium blinding.
Filter aids
42
Batch Filters
1. Plate and Frame Filters
2. Pressure Leaf Filters
Vertical metal-leaf filter
Horizontal metal-leaf filter
Stacked-disc filter
42
Continuous Filters
1. Rotary Vacuum Filters
2. Crossflow filtration
42
*used for the solid-liquid separation of various suspensions and has a wide range of applications.
*On an industrial scale the blank is one of the
cheapest filters per unit of filtering space and requires the least floor space, but it is intermittent in operation (a batch process) and there may be considerable wear of filter cloths as a
result of frequent dismantling.
*This type of filter is most suitable for fermentation broths with a low solids content and low resistance to filtration.
Plate and Frame Filters
43
* It is widely used as a 'polishing‘ device in breweries to filter out residual yeast cells following initial clarification by centrifugation or rotary vacuum filtration.
*It may also be used for collecting high value solids that would not justify the use of a continuous filter. Because of high labor costs and the time involved in dismantling, cleaning and reassembly, these filters should not be used when removing large quantities of worthless solids from a broth.
Plate and Frame Filters
44
*These filters incorporate a number of leaves, each consisting of a metal framework of grooved plates which is covered with a fine wire mesh, or occasionally a filter cloth and often precoated with a layer of cellulose fibres.
*The process slurry is fed into the filter which is operated under pressure or by suction with a vacuum pump.
*Because the filters are totally enclosed it is possible to sterilize them with steam.
*This type of filter is particularly suitable for 'polishing' large volumes of liquids with low solids content or small batch filtrations of valuable solids.
Pressure Leaf Filtration
45
*Use porous membranes to separate suspended
particles with diameter between 0.1 and 10 μm.
*operated at 0.2 to 3.5 bar pressure and typical value of fluz is above 200 L/m2/h
*It is most widely used for separation of suspended particulate matters, bacteria, fragmented cells or large colloids from solution.
Microfiltration
46
*The most widely used
process design in which the
entire fluid flow is forced
through the membrane under
pressure.
*As particles accumulate on
the membrane surface or in
its interior, the pressure
required to maintain the
required flow increases, until
at some point the membrane
must be replaced.
In-line filtration
47
*The feed solution is
circulated across the surface
of the filter, producing two
streams: a clean particle-free
permeate and a concentrated
retentate containing the
particles.
*The equipment required for
the Blank is more complex, but the
membrane lifetime is longer
than with in-line filtration.
Cross flow filtration
48
* A Blank has a pore size around0.01 μm.
* Microfiltration has a pore size around 0.1 micron, so when water undergoes microfiltration, many microorganisms are removed, but viruses remain in the water.
* Blank would remove these larger particles, may remove some viruses.
Ultrafiltration
49
*Reverse osmosis filters have a pore size around 0.0001 μm.
*After water passes through a Blank, it is essentially pure water.
* In addition to removing all organic molecules and viruses, it also removes most minerals that are present in the water.
* removes monovalent ions, which means that it desalinates the water.
Reverse osmosis
49
* has a pore size around 0.001 μm.
* removes most organic molecules,
nearly all viruses, most of the natural organic matter and a range of salts.
* removes divalent ions, which make
water hard, so Blank is often used to soften hard water.
Nanofiltration
50
is a technique used for the separation of
particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed.
*The particles are suspended in a liquid medium and placed in a centrifuge tube.
Centrifugation
51
The centrifuges used in harvesting fermentation broths are all operated on a
continuous or semicontinuous basis.
52
Types of Centrifuges:
1. Analytical
2. Preparative
53
*allows to analyze the mutual interactions between the subunits of multiprotein complexes and to unravel physico‐chemical properties like
the mass and size of macromolecules.
* Incorporate a scanning visible/ultraviolet light-based optical detection system for real-time monitoring of the sample’s progress during a spin.
Analytical
54
* often used for separating particles according to their densities, isolating and/or harvesting denser particles for collection in the pellet, and clarifying suspensions containing particles.
*Sometimes researchers also use preparative ultracentrifuges if they need the flexibility to change the type of rotor in the instrument.
*Preparative centrifuges can be equipped with a wide range of different rotor types, which can spin samples of different numbers, at different angles, and at different speeds
Preparative
55
RANGE OF CENTRIFUGES:
*Basket centrifuge (perforated-bowl basket centrifuge)
*Tubular-bowl centrifuge
*The solid-bowl scroll centrifuge (decanter centrifuge)
*Multichamber centrifuge
*Disc-bowl centrifuge
56
In SCP processes, Blank has been used as a flocculating agent since it can be used as a nutrient in medium recycle with considerable savings in water usage
phosphoric acid
57
The majority of flocculating agents currently in use are Blank, which act by charge neutralization and hydrophobic interactions to link cells to each other.
polyelectrolytes
58
Cell Disruption Methods
Physical methods
Chemical and biological methods
59
Physical methods
* Disruption using liquid shear
* Disruption using solid shear
* Agitation with abrasives
* Disruption using French press
* Disruption using ultrasonication
* Hydrodynamic cavitation
60
Chemical and biological methods
* Detergents
* Osmotic shock
* Alkali treatment
* Enzyme treatment
* Solvent
