Training Report On Commercial Production Of Biofertilizers

Submitted to:: Bio-tech Dept. JMIT, Radaur.

Submitted by:: Dinesh Kumar Bhardwaj Roll No.- 1208913 Biotech{5th sem.}

ACKNOWLEDGEMENT
While presenting this report, I owe my heartful thanks and gratitude to HINDUSTAN BIOENERGY LTD. for giving me this golden opportunity to work in their enriched and well equiped laboratories.

I am also very thankful to Mr. Sanjeev Chaudhary, Director, HBL, for allowing me to work with the company and take part in commercial work. the

Thanks to Dr. R.K. Shukla, Production Manager and Ms. Rachna Singh, our guide who made me understand things better.

I would also like to thank other Technician and Associates who helped me.

Dinesh Kumar Bhadwaj

CONTENTS
1..Introduction:: Biotech Park. Hindustan Bioenergy Limited. 2.. General Techniques Of Microbiology::
Cleaning of Glasswares. Sterilization of Glasswares. Media Preparation and Sterilization. Plate & Slant Preparation. Inoculation. Incubation and culture maintenance.

CFU Count. 3.. Introduction To Biofertilizers: What are Biofertilizers? Uses and Benefits of biofertilizers.

4.. Commercial Production of biofertilizers:-

Biotech Park
Bio-tech Parks are central government research institute which aims at attaining new heights in biotechnology research and shaping biotechnology for creation of wealth and ensuring security of food and health. There are even some private companies which are established within bio-tech park and works for mutual benefit. The park provides them space, water, electricity, allows them to use its technology, equipments and infrastructure and in return companies not only pays the park a huge amount as rent but also their production are directed by the park according to need of government and public.

Hindustan Bioenergy Limitied:From Nature, By Nature, To Nature

The company is in Public-Private Partnership with Bio-Tech Park, LUCKNOW.

Company has access to the infrastructure & facilities available at Biotech Park for the production of biofertilizers & Biopestisides.

Company also owns tissue culture lab for the production of Jatropa plant & Biodiesel which is at Kundanganj, about 70km from here.

Company deals in Bio Fertilizers, Organic Fertilizers & Crop Nutrition, Bio Pesticides & Bio Control Agents, Herbal or Botanical Crop Protectors, Soil Testing etc.

 General Techniques Of Microbiology:Microbiology::

It is the study of microorganism which are unicellular and too small to be seen with the naked eyes. They mainly includes bacteria, fungi,

Cleaning Of Glassware:Cleaning of glassware includes the following step:i. ii. Washing with in water with liquid detergent i.e {Tween-20} Acid wash Dip the glassware in water containing low conc. {3-4%} of strong acid {HCl or H2SO4}. Rinse with distilled water or alcohol to facilitated drying. Allow them to dry.

iii.
iv.

Sterilization of Glasswares::
Sterilization in general terms may be defined as the Destruction of all forms of life. But here we only consider the life of microbes. While working in Bio-Tech Lab. We generally use sterilized glassware & other apparatus to avoid any contamination. Generally Sterilization methods are divided into 2 categories:i. ii. Physical Methods Chemical Methods

Generally Sterilization methods::

Physical Methods::
Sterilization is done by using physical agents like heat, radiation, filters etc. These methods are generally used in Bio-Tech labs. Physical Methods have a major advantage that is they can be used for sterilization of not only glassware but also for sterilization of media. Examples:- Autoclave, Oven, UV Tubes, Laminar flow etc

i. Dry Heat Sterilization:It is one of the earliest form of sterilization. In this case microbes are destroyed by heat treatment. As the name indicates, dry heat utilize hot air that is either free from moisture or has very less of it. Dry heat sterilization process is a accomplished by conduction; which is a very slow process and it takes much longer for heat to reach microbes. Thus both time and temperature has to be increased. Generally we keep the glassware to be sterilized in hot air oven at 150C for 2 hrs.

ii. Moist Heat Sterilization:In this case sterilization is done by super-heated steam. Moist Heat is Better than Dry Heat as:i. ii. Steam is at higher temp. than hot dry air. Steam is more penetrating.

Autoclave Basic Principle:: When pressure {about 15 lbs} is applied to the boiling water, it increases the boiling point from 100 to 121C. Thus increasing the temperature & penetration power of steam. Generally used for glassware & even media.

iii.

Sterilization By UV radiation:This is the most commonly used method used for

room sterilization. This method is also quite often used for sterilization of glassware & even media. Best used for sterilization of heat sensitive materials. Basic principle:- UV radiations of wavelength 256nm are readily absorbed by Pyrimidines and undergoes diamer formation. Thus causing mutation which can be repaired. But the continuous exposure causes mutation that are lethal.

iv.

Flame:It is a kind of dry heat sterilization in which objects are heated under direct flame till it becomes red hot. Commonly used for sterilizing inoculation loops, metal spatulas, forceps etc. Sometimes metal instruments are dipped in ether or alcohol and then heated under the flame allowing the complete surface burn-off. Glassware {like slides, glass rod and mouths of test tubes, flasks, pipettes} are also sterilized by flame. This is done by passing it rapidly through the hot flame.

v.

Filtration:Filtration can be used for sterilization and filters are also used widely to avoid any contamination. There are certain fluids which cannot be subjected even to the moderate amount of heat{heat sensitive} without altering their nature. Such fluids can be sterilized by filtration. The best example of the air filtration is the use of cotton plugs in flask or test tubes. The cotton is porous enough to allow the necessary interchange of gases but will dont allow either dust or contaminating microbes to enter.

Chemical Methods::
Chemicals are used for sterilization but they have limited use in field of biotechnology. Chemicals are generally used for: Washing out vessels & Sterilizing the hands. Sterilizing of metal and some non-metal instruments. KMnO4 fumes are used for room sterilization.

Few examples:0.001% mercuric chloride, 1.5% formalin, 5% phenol, ethanol, ether, KMnO4 , formaldehyde etc are used.

Media Preparation::
Growth Media:Media is nothing but anything that provides source of energy & all the required nutrients in appropriate proportion and also provides appropriate pH for the GROWTH OF LIFE. It allows the microbial cells to grow and reproduce under favorable conditions.

Types of Media:According to different composition and different mode of action media can be classified as fallows:i) Complex Media: Rich in almost all different nutrients. Most of the microbes can grow on it. Exact composition is unknown hence called complex

ii) Selective Media:Suppresses the growth of unwanted microbes & also encourages the growth of desired microbes. For example:- If a microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance.

iii) Defined Media:Composed of pure ingredients in carefully measured concentration in distilled water.

iv) Differential Media:Distinguish colonies of specific microbes from others. It is also called indicator media indicators such as phenol red, methylene blue etc are added to it.

v) Enrichment Media:Increases the no. of desired microbe to detectable level without stimulating the rest of the bacterial population.

vi) Minimal Media: Contain the minimum possible nutrients for colony growth. Generally without amino acids. Used to grow "wild type" microorganisms

Procedure steps::
i. ii. Sterilize the media in autoclave, after that allow it to cool to about 50C. Now hold the flask in your right hand, agitating continuously to keep the temperature uniform and avoid partial solidification. iii. iv. v. vi. vii. viii. ix. x. Remove the cotton plug and flame the bottle mouth. Partially lift the cover of the first plate and Pour about 15-20 ml of media in each plate. Spread the liquid media on all the surface & close the plate cover. Repeat the same steps for the other Petri dishes. When done, wait until media solidifies without moving the plates. Turn the dishes upside down (media on top, cover on bottom). Incubate at 30C for 24 hours in a dry, dark, clean, dust-free condition. Check media surface for mold or bacteria growth, and discard contaminated plates.

Serial Dilution & Plate preparation:-

Procedure Steps::
i. Prepare the blanks by taking 9ml distilled water in test tubes and sterilizing test tubes in auto clave. ii. iii. iv. v. vi. vii. Mark the tubes as 1,2,3..and so on. Put 1ml of sample in 1st test tube. Then take 1ml from 1st tube & inoculate the 2nd tube. Repeat the same procedure for all tubes. Now inoculate agar petri plates taking sample from the tubes. Observe the growth after incubation and calculate CFU

Inoculation

CFU Count:Colony Forming Unit (CFU) is a measure or a count of viable microbial cell number. For convenience the results are given as:: CFU/ml (colony-forming units per milliliter) for liquids. CFU/g (colony-forming units per gram) for solids. For Example:: We have got 13 colonies formed in the plate which was inoculated with 8th dilution test tube. So in this case the CFU count will be = 13 X 10

Slant Preparation:-

i. ii. iii. iv. v. vi.

Take required no. of test tubes. Pour about 13ml of molten sterilized agar media in each of them. Put the tubes in slant position till the agar media solidifies. Incubate for overnight & next day check for contamination. Pick up a colony from petri plates and steak on slant culture. Incubate for overnight and check growth next day

Now Biofertilizers
Biofertilizers are any substance containing leaving microorganisms that helps plant to up take nutrients by their interactions in the rhizosphere when added to soil. They accelerate certain microbial process in the soil which extent availability of nutrients in a form which can easily be taken by plants. One of the major concerns in today's world is the pollution and contamination of soil . The use of chemical fertilizers and pesticides has caused tremendous harm to the environment. An answer to this is the biofertilizer, an environmentally friendly fertilizer.

Benefits::
Increases crop yield. Low cost. Non toxic. No harmful effect on soil and on the health of farmers. Renewable source. Maintains long term soil fertility. Suppress pathogenic soil organisms. Degrade toxic organic chemicals.

Microbes as biofertilizers::
Nitrogen fixing:: Rhizobium sp. Azotobacter sp. Azospirillum sp. Blue green algae. Azolla sp.

Phosphate Solubilizing:: Bacillus subtilis. Pseudomonas striata.

Silicates & Zinc solubilizers:: Bacillus sp.

Plant growth promoting:: Pseudomonas sp.

Commercial Production Of Biofertilizers

Upstream processing:Step 1:: Dealing with stock culture:Stock Culture are the improved stains of concerned microbes which are ordered from some high class microbiology labs. They are result of genitic engeneering and r-DNA technology hence somewhat costly & cannot be ordered daily.So what we do is we make some slant tubes and culture some plates from stock culture. Which can acts as stock culture for daily use. Step 2:: Preparing Mother Culture:Mother Culture are prepared on a small scale in 500 ml flasks. They are prepared to study cell growth & optimum growth condition of microbes. They are valid for 15 days. Following steps are followed:i. Media Preparation:- We prepare defined media for the specific microbe which we need to culture. ii. Media Sterilization:- In Autoclave at 121C and also under UV rays for 2 hrs. iii. iv. Inoculation:- with slant culture. Rotary shaker:- We place inoculated flasks on rotary shakes to provide agitating.

Step 3:: Preparing starter culture:Starter culture are prepared in the same way as mother culture but this time on somewhat large scale i.e. in 5 liters flasks. It act as inoculum for fermenter media. Following steps are followed:i. ii. iii. iv. Media Preparation:Media Sterilization:Inoculation:- with Mother Culture. Rotary shaker:- We place inoculated flasks on rotary shakes for 2-3 days.

Media composition:For phosphate solubilizing bacteria {1litre}: Glucose Ca3(PO4)2 (NH4) 2 SO4 KCl MgSO4.7H2O MnSO4 FeSO4 Yeast Extract ----- 10.0g ----- 05.0g ----- 00.5g ----- 00.2g ----- 00.1g ----- 0.06g ----- 0.03g ----- 00.5g

Distilled Water ----- 1000ml

Fermenter Basics:Fermenter or bio-reactor is any system or equipment that provides suitable environmental conditions for the growth of microbial or animal cell. The following condition can be changed and controlled: Ph Temperature Pressure Arriation rate Agitation rate

Fermenter Procedure:Here also the same procedure steps are followed but now in somewhat different way and quite large scale. Fermenter used is of 200 liters capacity. Following steps are followed for production of biofertilizers:i. Filling Fermenter Tank:Fill fermenter tank with 130 liters of water {about 65%}.

ii.

Adding Media Salt:Now add media salts and make up volume up to 150 liters {about 75%}. Turn on the Agitator for proper mixing, let it move for 2 hrs till mixed properly. Thus media is prepared.

iii.

Media Sterilization:Now as the media is in large quantity it can not be sterilized by conventional methods. So..... Fill the outer jacket of the fermenter with water. Fill it to about 40%. Now turn-on the jacket heater and let the temperature rise up to 121C. This will take about 4 hrs. Once temp. is attained, turn off the heaters. Now allow it to cool down to 30C. For this we will have to leave it for overnight.

If we are out of time and need it to be done quick, in that case we can pass cold water through outer jacket. Cold water supply at bottom, hot water outlet at top, Let it go till temp. falls down.

iv) Inoculation:Now inoculate with 30 liters of starter culture {about 15%}. This fills the fermenter upto 90%, the remaining 10% is kept empty keeping in mind the factors like proper mixing, form formation, air exchange. Generally it is advised to keep the fermenter 25% empty for the purpose of safety, but in companies profit matters, and even we should not forgot its INDIA. Procedure followed: Clean the feed vessel with alcohol. Keep the flame near feed vessel so that we could sterilize the neck of culture flask. Inoculate the fermenter with starter culture.

Once done close the flow and close the inlet pipe with cotton plugs.

v.

Arriation & Incubation:Pressure pump are connected to the spargers for arreation and then incubated for 1-3 days depending on the growth rate of microbes. Doors to fermenter room is closed with DO NOT DISTURB tag out.

Downstream processing:1. Sampling Out:As the small fermenter was kept in a room which can be sterilized thus sampling out is just taking out the culture through the tap of a tanker. But in case of the large fermenter, the full hall or shed could not be sterilized so sampling out has to be done in a proper way using stream for sterilizing pipes and then sterile air for cooling and then taking out the sample in the sterilized drum. 2. Quality Checking:The quality of the bio-fertilizers produced in fermenter is checked by using serial dilution and then finding out the cfu count of the product sample. The cfu count of the product should be atleast 2 x 10 the standards as set by ISO. 3. Charcoal Mixing:Charcoal is generally used as a carrier in the production of biofertilizers. We can use peat soil, mud, manure and soil mixture but charcoal is generally preferred as: Cost is cheaper. Easily available. High organic matter content. to meet

 Non toxic. Water holding capacity is more than 50%. Easy to process.

4.

Processing of carrier Material:a. Charcoal is powdered to fine powder. b. Mixed with calcium carbonate in ratio of 1:10 to neutralise acidic nature. c. Mixture is put into oven for sterilization and helping it lose humidity. d. Now charcoal is activated and mixed with broth culture obtained from the fermenter in a sterile metallic tray. e. Finally packed in polybags and sealed with electric sealer.

5. 6.

Filling & Packing Storing Or Dispatching:-

REFERENCES:-

www.biofertilizers.com www.molecularstation.com www.stackyard.com www.sigmaaldrich.com