Equipment for biofertilizer preparation

1. UNIVERSITY OF AGRICULTURAL SCIENCES ,BANGALORE
COLLEGE OF SERICULTURE, CHINTAMANI
EAM 422(0+10)- PRODUCTION TECHNOLOGY
FOR BIO-FERTILIZERS
TOPIC: LaboratoryEquipmentsused in mass
productionof biofertilizers
Submitted to,
DR. Dakshayini.G
Dept Of Microbiology
COS, Chintamani
Submitted by,
Amina Aftab
ALC9004
IV Bsc(hons) Agriculture

2. The main equipments required for mass
production of bio-fertilizers are listed as
follows:
•Autoclave
•Rotary Shaker
•Fermentors
•Laminar Air Flow
•BOD Incubator
•Hot Air Oven
•Incubator
•Refrigerator
•Microscope
•Weighing Balance
•pH meter
•Colony Counter
•Glassware as needed. Conical flasks are major requirement
•DistillerWater Unit/ Demineralization Unit
•Sealing Machine

3. AUTOCLAVE
• An autoclave is a machine that provides a physical methodof
sterilization by killing bacteria, viruses, and even spores present
in the material put inside of the vessel using steam under
pressure. Autoclave sterilizes the materials by heating them up
to a particular temperature for a specific period of time.
• Principle: The autoclave works on the principle of moist heat
sterilization where steam under pressure is used to sterilize the
material present inside the chamber. The high pressure
increases the boiling point of water and thus helps achieve a
higher temperature for sterilization.

4. Autoclave Parts/ Components

5. a. Pressure Chamber
The pressure chamber is the main component of a steam autoclave
consisting of an inner chamber and an outer jacket.
The inner chamber is made up of stainless steel or gunmetal, which is
present inside the out chamber made up of an iron case.
b. Lid/ Door
The next important component of an autoclaveis the lid or door of the
autoclave.
The purpose of the lid is to seal off the outside atmosphere and create
a sterilizedcondition on ht inside of the autoclave.
Pressure gauge
A pressure gauge is present on the lid of the autoclaveto indicate the pressure
created in the autoclaveduring sterilization.
Pressure releasing unit/ Whistle
A whistleis present on the lid of the autoclave is the same as that of the
pressure cooker.

6. c. Steam generator/ Electrical heater
An electrical steam generator or boiler is present underneath the chamber
that uses an electric heating system to heat the water and generate steam in
the inner and the outer chamber.
The level of water present in the inner chamber is vital as if the water is not
sufficient;there are chances of the burning of the heating system.
Similarly,if the water is more than necessary, it might interfere with the
trays and other components present inside the chamber.
d. Vacuum generator (if applicable)
In some types of autoclaves,a separate vacuum generator is present which
pulls out the air from the inside of the chamber to create a vacuum inside
the chamber.
The presence of some air pockets inside the chamber might support the
growth of different microorganisms. This is why the vacuum chamber is an
importantcomponent of an autoclave.
e. Wastewater cooler
Many autoclaves are provided with a system to cool the effluent before it
enters the draining pipes.
This system prevents any damage to the drainage pipe due to the boiling
water being sent out of the autoclave.

7. Autoclave invented by: Charles Chamberland
Principle: Moist heat sterilization
Uses of Autoclave
•They are used to decontaminate
specific biological waste and
sterilize media, instruments, and
labware.
•In medical labs, autoclaves are
used to sterilize medical
equipment, glassware, surgical
equipment, and medical wastes.
•Similarly, autoclaves are used for
the sterilization of culture media,
autoclavable containers, plastic
tubes, and pipette tips.

8. 2.HOT AIR OVEN
•Hot air ovens are electrical devices which use dry heat to sterilize. They were
originally developed by Louis Pasteur. Generally, they use a thermostat to control the
temperature.
•To destroy microorganisms and bacterial spores, a hot air oven provides extremely
high temperatures over several hours.
•The widely used temperature-time relationship in hot air ovens to destroy
microorganisms are 170 degrees Celsius for 30 minutes, 160 degrees Celsius for 60
minutes, and 150 degrees Celsius for 150 minutes.
Working Principle of Hot air oven/Hot air oven principle
Utilizing convection, conduction, and radiation, a hot air oven operates
according to the dry air sterilisation procedure. The heating components
heat the chamber’s air, which can be evenly circulated with the use of
fans to expose sample surfaces to hot, dry air. This exposure causes the
external surface of an object to heat up, and the conduction mechanism
transfers the heat to the object’s core. Similarly, heat causes
microorganisms’ internal water to evaporate, resulting in oxidative
damage to cellular components, denaturation of proteins, a toxic
consequence of high electrolyte levels, and, finally,death.

9. What can be Sterilized by using a Hot Air Oven?
• Glassware: Glassware, such as test tubes
and beakers, can be sterilized in a hot air
oven.
• Laboratory equipment: Laboratory
equipment, such as pipettes and Petri
dishes, can be sterilizedin a hot air oven.
• Medical instruments: Medical
instruments, such as scalpels and forceps,
can be sterilized in a hot air oven.
• Fabric: Fabric, such as lab coats and
towels, can be sterilized in a hot air oven.
• Chemicals: Some chemicals can be
sterilized in a hot air oven by heating them
to a high enough temperature to kill any
microorganismspresent.

10. Advantages of dry heat sterilization
• A dry heat cabinet is easy to install and has relatively low operating costs;
• It penetrates materials
• It is nontoxic and does not harm the environment;
• And it is noncorrosive for metal and sharp instruments.
Disadvantages for dry heat sterilization
• Time-consuming method because of a slow rate of heat penetration and
microbial killing.
• High temperatures are not suitable for most materials e.g. plastic and rubber
items cannot be dry-heat sterilized because temperatures used (160–170°C)
are too high for these materials.
• The time and temperature required will vary for different substances and
overexposure may ruin some substances.

11. LAMINAR AIR FLOW
• A Laminar flow hood/cabinet is an enclosed
workstation that is used to create a contamination-
free work environment through filters to capture
all the particles entering the cabinet.
• These cabinets are designed to protect the work
from the environment and are most useful for the
aseptic distribution of specific media and plate
pouring.
• Laminar flow cabinets are similar to biosafety
cabinets with the only difference being that in
laminar flow cabinets the effluent air is drawn into
the face of the user.

12. Components/ Parts of Laminar Air Flow
1. Cabinet
• The cabinet is made up of stainless steel with less or no gaps or joints preventing the collection
of spores.
• The cabinet provides insulation to the inner environment created inside the laminar flow and
protects it from the outside environment.
• The front of the cabinet is provided with a glass shield which in some laminar cabinets opens
entirely or in some has two openings for the user’s hands to enter the cabinet.
2. Working station
• A flat working station is present inside the cabinet for all the processes to be taken place.
• Culture plates, burner and loops are all placed on the working station where the operation takes
place.
• The worktop is also made up of stainless steel to prevent rusting.
3. Filter pad/ Pre-filter
• A filter pad is present on the top of the cabinet through which the air passes into the cabinet.
• The filter pad traps dust particles and some microbes from entering the working environment
within the cabinet.
4. Fan/ Blower
• A fan is present below the filter pad that sucks in the air and moves it around in the cabinet.
• The fan also allows the movement of air towards the HEPA filter sp that the remaining
microbes become trapped while passing through the filter.

13. 5. UV lamp
• Some laminar flow hoods might have a UV germicidal lamp that sterilizes the interior
of the cabinet and contents before the operation.
• The UV lamp is to be turned on 15 minutes before the operation to prevent the
exposure of UV to the body surface of the user.
6. Fluorescent lamp
• Florescent light is placed inside the cabinet to provide proper light during the
operation.
7. HEPA filter
• The High-efficiency particulate air filter is present within the cabinet that makes the
environment more sterile for the operation.
• The pre-filtered air passes through the filter which traps fungi, bacteria and other dust
particles.
• The filter ensures a sterile condition inside the cabinet, thus reducing the chances of
contamination.
The principle of laminar flow cabinet
is based on the laminar flow of air
through the cabinet.
The device works by the use of
inwards flow of air through one or
more HEPA filters to create a
particulate-free environment.
The air is taken through a filtration
system and then exhausted across the
work surface as a part of the laminar
flow of the air.

14. Uses of Laminar flow hood
• Laminar flow cabinets are used in laboratories for contamination
sensitive processes like plant tissue culture.
• Other laboratories processes like media plate preparation and culture of
organisms can be performed inside the cabinet.
• Operations of particle sensitive electronic devices are performed inside
the cabinet.
• In the pharmaceutical industries, drug preparation techniques are also
performed inside the cabinet to ensure a particulate-free environment
during the operations.
Precautions
While operating the laminar airflow, the following things should be
considered:
•The laminar flow cabinet should be sterilizedwith the UV light before and
after the operation.
•The UV light and airflow should not be used at the same time.
•No operations should be carried out when the UV light is switched on.
•The operator should be dressed in lab coats and long gloves.
•The working bench, glass shield, and other components present inside the
cabinet should be sterilized before and after the completion of work.

15. BOD INCUBATOR
• A BOD (biological oxygen demand)
incubator is a device used to grow and
maintain microbiological cultures or cell
cultures.
• The incubator maintains optimal
temperature, humidity and other
conditions such as oxygen content of the
atmosphere inside.
• It is also known as low temperature
incubator or refrigerated incubator
because it is made with temperature range
between 5°C to 80°C or with cooling and
heating functions under one unit.

16. Components of BOD incubator
Temperature
• The temperature range of a BOD incubator is 5°C to 60°C.
• It contains a digital PID controller supported by a PT100 sensor to provide the
temperature accurately and uniformly.
Illumination
• It has a fluorescent or LED light, which is door operated.
• It also contains 3 tube lights on the door with a digital cyclic timer. It controls the
illumination 24×7 weekly.
Cooling
• It has a CFC free refrigeration system for cooling temperature under BOD
Incubator.
Heating
• It contains ISI mark U-shaped tubular air heaters which help in heating.
Trays
• It contains 2 to 5 removable trays which are made of steel wire mesh cable.
• These are removable for cleaning purposes and height-adjustable.
Air circulation
• It contains axial fans or a motorized blower system.

17. Construction
• It is a double-walled incubator consist of exterior and interior walls.
• The exterior wall is consists of thick mild steel and powder coated.
• The interior wall is consists of stainless steel.
Applications
1. Many industries use BOD
incubators such as
pharmaceutical, agriculture,
beverages and research
laboratories.
2. In manufacturers, it is employed in
waste processing plants to define
the proformance of the treatment
system.
3. BOD incubator is employed in
liquors to distinguish the
nourishment in intendedworking
situation
4. It is employed in the farming
industry to define the
germination of anaerobic
bacteria.

18. COLONY COUNTER
• A colony counter is an equipment used to
count colonies of microorganisms
growing on agar plates.
• There are numerous types of colony
counters available for counting bacteria
and yeast colonies rapidly and precisely.
• Some of these colony counters are
manually operated, while others are
automatically operated.
• The colony counter is used
for microbiology applications for fast and
accurate counting of bacterial and mould
colonies.
Manual Colony Counter
Automated Colony Counter

19. BIOREACTORS
• A bioreactor is a type of fermentation vessel that is used for the
production of various chemicals and biological reactions.
• It is a closed container with adequate arrangement for aeration,
agitation, temperature and pH control, and drain or overflow vent
to remove the waste biomass of cultured microorganisms along with
their products.
A bioreactor should provide for the following:
• Agitation (for mixing of cells and medium),
• Aeration (aerobic fermentors); for O2 supply,
• Regulation of factors like temperature, pH, pressure,
aeration etc
• Sterilization and maintenance of sterility, and
• Withdrawal of cells/medium

20. Bioreactor Design
• The design and mode of operation of a bioreactor are based on the
production of an organism, optimum conditions required for
desired product formation, product value, and its scale of
production.
• A good bioreactor design will help to improve productivity and
provide higher quality products at lower prices.
• A bioreactor is a device that consists of various features such as an
agitator system, an oxygen delivery system, a foam control system,
and a variety of other systems such as temperature & pH control
system, sampling ports, cleaning, and sterilization system, and
lines for charging & emptying the reactor.
• The material used for the construction of a bioreactor must have
the following important properties:
– It should not be corrosive.
– It should not add any toxic substances to the fermentation media.
– It should tolerate the steam sterilization process.
– It should be able to tolerate high pressureand resist pH changes.

21. Parts Of Bioreactor
1. Fermenter Vessel
2. Heating and Cooling Apparatus
3. Aeration System
4. Sealing Assembly
5. Baffles
6. Impeller
7. Sparger
8. Feed Ports
9. Foam-Control
10. Valves
11. Controlling Devices for Environmental
Factors
12. Use of Computer in Fermenter

22. Types and Applications Of Biofertilizers
Types Application
Continuous Stirred Tank Bioreactors Production of the primary metabolites,
enzymes and amino acids.
Bubble column bioreactors cultivation ofherring-sensitive organisms. E.g.
Plant cells and mould
Air-lift bioreactors methanol production,waste water treatment,
single-cell protein production.
Packed Bed Reactors Used is waste water treatment.
Fluidized Bed Bioreactor The food processing industry
Photobioreactor The main applicationsof photobioreactorsare
in photosyntheticprocesses, involving
vegetablebiomass growth or microalgae
growth under restricted conditions.
Membrane Bioreactor widely used in treatment of waste waterfrom
several sources.

23. ROTARY SHAKER
• Rotary shaker is laboratory equipment for mixing and growing chemicals,
solvents, and other laboratory samples.
Rotary shaker function
1. To make the solution even out
• In general, whatever the type of rotary shaker, the main function is to oscillate
and mix samples at a certain temperature and speed, so that the solution is evenly
distributed.
In addition, in biochemical research or experiments, rotary shakers are used for
the culture of microorganisms, such as bacteria in liquid media.
2. Homogenize a solution of high viscosity
3. Homogenize nutrients
4. Samples can be homogenized at low temperatures

24. REFRIGERATOR
• Refrigeration slows microbial growth; freezing
stops growth, killing some organisms.
Laboratory and medical specimens may be
frozen on dry ice or at ultra-low temperatures
for storage and transport.
• Laboratory refrigerators are distinct from
regular refrigerators used in homes and
restaurants because they must be 100 percent
hygienic and 100 percent dependable.
• It facilitates the provision of correct
temperature monitoring and alarm, which
prevents temperature swinging, ensuring the
safety of biological materials and vaccines.
• It promotes better airflow (ventilation) through
powerful air circulation systems with air cooling
vents in refrigerators and freezers.
• It prevents or stops biological contamination in
laboratories.

25. pH Meter
• A pH meter is an instrument used to
measure hydrogen ion activity in solutions -
in other words, this instrument measures
acidity/alkalinity of a solution. The degree
of hydrogen ion activity is ultimately
expressed as pH level, which generally
ranges from 1 to 14.
• Microorganisms constitute a broad group of
lifeforms, where some strains are tolerant to
wide pH ranges and others require narrow
ranges. Generally, microbes prefer an
optimal pH value, where changing the pH
of the microbial environment reduces
metabolism and viability.

26. Weighing Balance
• Weighing is one of the most common
tasks in the microbiology laboratory.
• Microbiology laboratory weighing
balance mainly used for media
preparation,micro pipette calibration
and product testing purpose.
• Advanced micro,semi-micro,analytical
and precision balances have now been
perfected to such a degree that, in
general, no special weighing rooms are
needed.

27. COMMON GLASSWARES
Test tube, culture tube and screw-cappedtubes
• These glasses are made of glass with one
side being closed and the other open.
• It is known as a test tube if its side wall
curves outwardly from the end; it is also
known as a culture tube. A screw-capped
tube is characterizedby a tube with a side
wall that has screws so that he can be fitted
e.g. : a screw-capped tube
• They are used in microbiological
laboratories.
• Test tubes are used to test chemicals like
pH. Culture tubes can be used to prepare
agar slants or purify microorganisms.The
open end of the tube is filled with a non-
absorbent cotton plug.
• Sometimes,the microorganisms can be
purified and kept in screw-capped tubes.

28. Petridish
• This dish was created by R.J. Petri (a
student of Robert Koch, a renowned
bacteriologist) for the first time.
• It is made up of two small glass dishes: the
lid or upper half and the bottom or lower
half.
• These dishes can be used in any
microbiological laboratory to isolate and
cultivate different types of microorganisms.
Pipette
•Without pipette, the transfer of solutionfrom
one location to another would be messy and
unsafe.
•A pipette is used to very accurately displace a
volume if liquid. Pipettes can suspend volumes
from 1 µl (= 0,001 ml) accurately.
•They are therefore irreplaceableand of crucial
importance in a lab.

29. Volumetric flasks
• It is used for preparing solution of
exact strength.
• The upper portion is narrow and
cylindrical, and marked with a
point. This marks denotes the
water level that must be
maintained at this point.
• The lower half of the body is
round and voluminous.
• Its flat base allows it to be placed
properly on the surface

30. Glass Spreader
• You can make a glass spreader by
benting a piece of glass rod into an L-
shaped shape.
• It is used for spreading microorganisms
evenly on agar surfaces in liquid
medium.
• The long arm is held in your hand,
while the small arm is flame-sterilized
and placed on agar.

31. Bunsen Burner
• A Bunsen burner is a source of open flame
that is used to sterilize loops and needles, as
well as flaming the lips of test tubes during
inoculations.
Inoculating loops and inoculating needles
Inoculating loops and needles are used to
transfer bacteria into and from culture media.
Inoculating loops have a loop at the end,
while inoculating needles end in a point.
Inoculating loops are the most common
method of transferring bacteria.
Inoculating needles are used when stabbing
into a medium during specific inoculation
procedures, or when it is necessary to pick up
a small amount of bacteria from one colony
on an agar plate without contacting bacteria in
other colonies.

32. CULTURE MEDIA
• A culture medium is
essentially composed of basic
elements (water, nutrients), to
which must be added different
growth factors that will be
specific to each bacterium and
necessary for their growth.
• Nutrient broths and agar plates
are the most typical growth
media for microorganisms.
• Culture media is a gel or liquid
that contains nutrients and is
used to grow bacteria or
microorganisms.

33. Mother Culture
• A pure growth of any organism
on a small scale is called as a
mother culture. Mother culture is
always prepared in a conical flask
of 500 or 1000 ml. Capacity and
then this mother culture is used
for further production.
Sub Culture
A subcultureis either a new cell culture
or a microbiological culture made by
transferring some or all cells from a
previous culture to fresh growth
medium.This action is called
subculturing or passaging the cells.